Metamorphosis in the Cnidaria1

Home , Eirene (genus), Hydractinia, Hydractinia echinata, Planula

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1755

REVIEW/SYNTHÈSE

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 substrate- borne 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 immobi- les 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 cer- tains anthozoaires (Nematostella, Acropora), scyphozoaires (Cassiopea) et hydrozoaires (Coryne, Proboscidactyla, Hy- dractinia). 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 sensi- bles 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 capa- bles 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] Müller and Leitz 1771

Introduction during their embryonic phase of life cnidarians develop a planula larva which settles on a substrate and transforms Cnidarians are primarily members of communities that oc- into a benthic phenotype or morph known as a polyp. This cupy benthic habitats. “Primarily” in this context means that 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. 1This 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. 2Corresponding author (e-mail: [email protected]).

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ancient precursors in the evolution of the phylum (Miller Fig. 1. Paradigm of a (metagenetic) cnidarian life cycle: Aurelia and Ball 2000). Even if the life cycle of a species includes a aurita (Scyphozoa). 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. Remark- ably, 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 infor- mation 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 pat- ited. It consists of mechanosensitive or chemosensitive neuro- tern of development and behaviour (Hofmann et al. 1996). sensory 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 General ecophysiological aspects not enable them to locate an adequate habitat from a distance; this is discussed in the following section. As a rule, How do cnidarians find and select an appropriate habitat? the larvae of sedentary organisms depend on a hierarchy of The life cycle of all sedentary organisms includes a free- key stimuli that are indicative of their adult environment and living stage, which has two predominant assignments: (1) to lead them to their destination stage by stage. In terms of be- exploit a scattered or transient ecological niche and (2) to haviour, larvae often display searching activities until they promote genetic exchange throughout the various popula- are presented with a specific stimulus that triggers settle- tions of a given species. Colonists arriving at appropriate ment and metamorphosis. sites from different home populations will most probably contribute different alleles to the common gene pool of the Are navigation and chemotaxis possible from a distance? colony, thus augmenting its genetic diversity and flexibility Chemotaxis from a distance, guided by soluble and diffus- and enhancing its chances of long-term survival. ing target-borne molecules under natural conditions of water In cnidarians the task of prospecting for and identifying a turbulence, is probably not a sufficiently reliable means of congenial habitat is not assigned to the elaborate planktonic detecting a distant preferred substrate or precisely locating a swimming phase known as the medusa or jellyfish, a morph specific site in the habitat, such as a particular species of that is lacking in the Anthozoa anyway, but to the tiny planula alga. The presence of abundant diffusible molecules such as + larvae, that is, those multicellular spindle-like or elliptical NH3/NH4 ,H2S, amino acids, and other conventional organic bodies which in sexual reproduction arise from fertilized compounds can indicate a favourable or inappropriate large- eggs (Fig. 1; see also Fig. 5); or the task is assigned to scale habitat like a mangrove swamp. But more specific planula-like buds called propagules, which are asexually diffusible factors are likely to be present in perceptible quan- produced and released from polyps or, in rare instances tities only in the viscous boundary layer adjacent to the sub- (trachyline Hydrozoa), from medusae. Even when the strate or directly on its surface. Moreover, the minuscule metagenetic life cycle has provided the species with a large cnidarian larvae are subjected to flow characterized by low medusa capable of brooding the eggs throughout develop- Reynolds numbers and are more apt to be carried along by ment until the settling stage, as in the genus Cassiopea (see the mass of water than to travel through it. below), the larvae are eventually set free and it is their job to find a suitable substrate on which to settle. Except in sea How do pelagic larvae find a substrate? anemones, which remain mobile, the polyp arising from the The task of prospecting for a congenial substrate appears larva is permanently bound to a substrate and cannot correct to present different levels of difficulty for pelagic (plank- a wrong choice. Careful habitat selection, therefore, is essen- tonic) larvae compared with benthic larvae, since the latter tial for the survival of a population. But how can the tiny move over the sea floor. Pelagic larvae, or the medusae and larvae carry out such a task? Their sensory equipment is lim- jellyfish that release them into the expanse of the ocean, are

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Fig. 2. Patterns of vertical movement of planktonic larvae in the Fig. 3. Effect of tension of wetting on the attachment of a larva. water column.

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 Levels of specificity of settlement: a case to travel through a large column of water until they reach study the bottom. However, pelagic larvae commonly move up and down the water column in a daily rhythm, presumably As a rule, different species have different final destina- in response to an internal circadian clock and guided by tions. Patterns of abundance may reflect both passive deposi- light and (or) gravity, or they are passively shifted by ther- tion of larvae that sink as particles under the influence of mally driven convections that arise and fade daily (Fig. 2). local hydrodynamic conditions and successful, active sub- At the end of their sinking phase the larvae may approach strate selection. A substrate covering areas whose size sur- the bottom, so they have the opportunity to explore the envi- passes the average spatial scale of passive larval dispersal ronment near the sea floor at daily intervals. If a larva enters could be colonized simply by random deposition of larvae. an appropriate habitat, it may gradually be guided to an ap- The more specific the destination, the more specific the cues propriate site by a cascade of cues. The hierarchy of cues that are needed in order to restrict settlement to particular may include unspecific chemical or physical parameters, sites within the habitat. The following examples represent + such as the NH3/NH4 content or viscosity of the water, increasing demands of specificity. microcirculation properties close to the surface of solids, surface energies such as the tension of wetting, the thermal Nematostella vectensis: a soft-bottom-colonizing cnidarian capacity of the substrate, and other parameters indicative of Nematostella vectensis is a small euryhaline sea anemone the presence of a particular substrate and its quality (e.g., found in estuaries along the European and American coasts soft versus hard). At this point at the latest, pelagic and ben- of the North Atlantic Ocean, along the shores of the Gulf of thic larvae are now confronted with the following problem, Mexico, and along the Pacific coast of Mexico (Hand and which is common to all larvae below a critical size. Uhlinger 1992 and references therein). The sea anemone occurs in soft sediments, in plant debris, and among living How do larvae establish physical contact with a substrate? plants in permanent pools and tidal creeks in salt marshes. In Tiny larvae can find it difficult to establish intimate con- the laboratory the animals are sexually active throughout the tact with a solid surface. They have to overcome several year, showing no sign of seasonality in their reproduction, physical barriers such as shear stress, forces of surface ten- although in nature reproduction may depend on favourable sion, and repulsive electrostatic potentials. Of particular im- external conditions. Male and female sea anemones prefer- portance is the liquid/solid-surface energy known as tension entially spawn in the early evening. The eggs are discharged of wetting (Müller et al. 1976). If the surface of the substrate in masses embedded in gelatinous material. They develop and the surface of the larva both display hydrophilic proper- into planulae, which are equipped with an apical tuft of long ties, high capillary forces strongly bind a water film at the cilia. Most likely this tuft is a sensory organ. However, meta- larva–substrate interface, and this film is not easily dis- morphosis of the planulae appears not to depend on specific lodged by the larva. On the other hand, if both the surface of environmental cues and is even initiated before settlement. the substrate and the surface of the larva are hydrophobic, In the laboratory the pear-shaped larvae have already devel- the larva is attracted to the substrate as a a drop of oil would oped tentacles by day 5 post insemination, i.e., before settle- be (Fig. 3). Adhesion through holdfasts and cements is also ment; they cease swimming, sink to the bottom, and strongly influenced by the physical properties of the substrate. complete their transformation into juveniles about 1 mm in This applies not only to animal larvae but also to microbes. length. As the larvae spend only a short time in the plankton, Only in recent years has it been recognised (by botanists us- their dispersal is mostly restricted to the area of their birth. ing a scanning electron microscope to look at the ever-clean Moreover, the small sea anemone that arises from the larva

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is not bound to a particular place. During the first few days low tropical lagoons and mangrove swamps, often lying upside after metamorphosis, the juveniles glide over the bottom down on a sandy bottom instead of swimming around like with the aboral end forward. (After a few days the direction other jellyfish. The adult females incorporate sperm released of movement reverses.) If the moving juveniles find a soft into the surrounding water by the males. They envelop the in- sediment, they burrow into it by digging their peduncle into ternally fertilized eggs in mucus and wrap this mass around the the sediment. The independence of metamorphosis from spe- base of sex-specific vesicles, thus protecting the embryos until cific inducing external cues reflects the wide distribution of the ciliated planula larvae hatch from the egg envelopes. In ad- the metamorphosed animals on the substrate, and their mo- dition to sexually generated planulae, Cassiopea also produces bility; they remain able to change their position if local con- planula-like swimming bodies by asexual budding. Both types ditions prove not to be favourable. In favourable places the of larvae, whether produced sexually or asexually, have to find animals start cloning themselves by transverse fission. Thus, a hard substrate on which to settle and transform into a polyp a pond can be colonized quickly and the quantity of eggs (called a scyphistoma). There is experimental evidence that the produced rises. larvae avoid settling on clean, sterile substrata. Instead, they attach only to substrata covered with a microbial film to undergo The coral Acropora millepora: a hard-bottom dweller metamorphosis (Hofmann et al. 1996). The presence of a sur- In marine environments, hard substrata are much less ex- face film of microorganisms has long been recognised as a pre- tensive, and far more patchily distributed, than sedimentary requisite for the settlement of many fouling invertebrates deposits. Therefore, selective pressure for a high degree of (Pawlik 1992 and references therein). The supporting effect of site-specific settlement among hard-bottom dwellers is more microbial films has commonly been attributed to their physical intense. properties. The bio-organic film may affect patterns of larval Besides Nematostella, the coral Acropora millepora has settlement by virtue of the altered tension of wetting it confers been proposed as a new model organism representing the on a hard substrate. However, bacteria in the environment also class Anthozoa in particular and the phylum Cnidaria in gen- provide soluble cues: by decomposing mangrove leaves they eral (Miller and Ball 2000). In the area of the Great Barrier liberate proline-rich peptides that cause the larvae to terminate Reef, many corals, including A. millepora, practice synchro- larval life and enter metamorphosis (Fitt and Hofmann 1985; nous mass spawning once a year. The conventional embry- Fitt et al. 1987; Fleck and Fitt 1999; Fleck et al. 1999). onic development of the floating eggs leads to spindle-shaped Preference for a substrate covered with a bio-organic film planula larvae, the typical dispersal stage for most sedentary is also displayed by other scyphozoan planulae. The planulae cnidarians. The planulae contain large amounts of lipids, en- of Cyanea have been reported to settle on aged mollusc abling them to remain in the water column for months. The shells rather than on freshly vacated shells, and this prefer- larvae are passively dispersed by currents and convections in ence has been attributed to the decreased wettability of film- the water column but also display active swimming behav- covered surfaces (Brewer 1984). However, as will be shown iour that is driven by cilia. They have a well-developed ner- with larvae of Hydractinia (“Metamorphosis of Hydractinia, vous system, a mouth opening into a gastrovascular cavity, the best studied cnidarian model organism” below), the in- and nematocysts. fluence of substrate-bound bacteria can be more specific and When the planulae are ready to settle, they sink to the bot- indicative of a particular substrate. tom, showing a characteristic rotating swimming pattern as they repeatedly test the substrate for a place to settle. Only Proboscidactyla flavicirrata, Coryne uchidae, and others: after having settled do the larvae undergo metamorphosis living on biogenic substrata and form tentacles. If the preferred substrate is biogenic, the cues mediating Since the planulae have been reported to test the substrate specificity or enhancement of settlement are believed to be actively and repeatedly, we can infer that they are probably ex- chemical in nature. However, in no case has the chemical ploring it in search of some characteristic properties. Is one of stimulus been identified with certainty, although pioneering these properties the presence of substrate-bound bacteria, as investigations made in Japan (Nishihira 1968; Kato et al. in case of the hydrozoan genus Hydractinia (described below)? 1975) were promising. In recent experimental investigations it was concluded that The hydroid Coryne uchidai settles on brown algae of the bacteria are among the sources of inductive signals (Negri et al. family that includes the genus Sargassum. Boiled aqueous ex- 2001). tracts (Nishihira 1968) as well as hexane extracts from dried In future studies, attention should also be given to a physi- Sargassum tortile (Kato et al. 1975) caused larvae to cease cal phenomenon not hitherto taken into account. In areas swimming, and a varying fraction of the motionless larvae un- with hard substrata, particularly along rocky shorelines and derwent metamorphosis Several diterpenoid chromanols have reefs, surf produces air bubbles and foam, and such condi- been isolated, among them δ-tocotrienol epoxide (Fig. 4), tions have been found to induce sinking and the onset of which caused larvae to metamorphose. However, only a few metamorphosis in the scyphozoan Aurelia aurita (Kroiher larvae were available for bioassays, and it is not known and Berking 1999). whether the identified diterpenoids are exposed on the surface of the algae. Any chemical that interferes with internal Another case: Cassiopea and bioorganic cues triggering mechanisms could initiate metamorphosis without Cassiopea (e.g., C. andromeda, C. xamachana) is a jellyfish being the natural inducer. This became evident in our studies genus that differs in its habits from other genera of the typi- on Hydractinia: the planulae of this species can be induced cally holopelagic scyphomedusae, as it prefers to live in shal- to metamorphose with the use of tumour-promoting phorbol

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Fig. 4. Chemical structure of a substance derived from an alga channels formed by tubular stolons that run along the sub- (Sargassum sp.), δ-tocotrienol epoxide, which causes settlement strate. Colonies grow by peripheral extension of the tubular and metamorphosis of the planulae of the hydroid Coryne stolons, which ramify and anastomose, forming a dense net- uchidae. work. 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). esters (Müller 1985), and these compounds are derived not from marine plants but from terrestrial plants (of the family Sexual reproduction Euphorbiaceae). Phorbol esters are highly active without be- In Hydractinia colonies the sexes are separate. Under stan- ing natural inducers. The particular processes that are acti- dard conditions in the laboratory, mature colonies spawn vated by phorbol esters will be discussed in the following synchronously and almost daily 1–2 h after the onset of illu- section (“Metamorphosis of Hydractinia, the best studied mination in the morning. The gametes are released into the cnidarian model organism”). surrounding seawater, where fertilization occurs. After 3 days Epiphytic associations are common in several species of ma- the fertilized eggs develop into planulae, which are compe- rine organisms. For instance, the scleractinian coral Agaricia tent to metamorphose. However, in clean water and under humilis prefers to settle on certain crustose coralline algae. A sterile conditions the larvae do not enter metamorphosis. In compound has been extracted from these algae that induces the the laboratory they can be kept (at 6°C) for months until settlement and metamorphosis of A. humilis larvae (Morse et they starve to death, but they will not undergo metamorphosis al. 1988, 1994; Morse and Morse 1991). The compound ap- without being stimulated by natural or artificial key stimuli pears to be a macromolecule containing sulfated glucosamino- or chemical inducers. In nature, the best place for Hydractinia glycan. planulae to select for settling is a shell inhabited by a hermit Proboscidactyla flavicirrata is found solely on the tube crab. rims of sabellid polychaetes. The planulae establish prelimi- nary physical attachment to a substrate by extruding the sticky threads of nematocysts. Nematocysts of the particular The benefits of a mobile home type borne by the planulae of this species were specifically On shoals and mud flats exposed to strong tides with large stimulated to discharge on contact with the feeding append- lifts, a mobile home carried by a hermit crab provides many ages or body surface of sabellids, resulting in larval attach- benefits for an epiphytic symbiont such as Hydractinia, while ment to the worm (Donaldson 1974). Threads of particular the crab benefits because the dangerous nematocysts of its nematocysts (atrichous isorhizas) are also used by the actinula symbiotic partner, the hydroid, discourage its potential pred- larvae of Tubularia mesembryanthemum (Yamashita et al. ators (Brooks and Gwaltney 1993 and references therein). 1998) and planulae of Hydractinia (below) to attach to a The following list summarizes the unpublished observations solid surface. of one of the present authors (W.A.M.). • The hermit crab frequently roams about in search of food. Metamorphosis of Hydractinia, the best While the shell is dragged along the bottom, the large feed- studied cnidarian model organism ing polyps found along the lower edge of the shell’s mouth can fish for and collect the many small nematodes, annelids, The organism and crustaceans that live in the superficial layers of sandy The colonial marine hydroid Hydractinia has in recent bottoms. The crab also ferrets out small animals hidden in years progessively acquired the status of a cnidarian model the grooves of hard bottoms or in bunches of algae. organism (Leitz 1998; Frank et al. 2001). Hydractinia occurs • Shells exposed to tidal currents in sandy areas are fre- in the North Atlantic Ocean as two sibling species, Hydractinia quently covered with sand. Crabs can quickly dig them- echinata along the coasts of northern Europe and Hydractinia selves and their home out. symbiolongicarpus along the coast of North America (Buss and Yund 1989). The large-scale habitat of these two closely • On shoals, particularly around the North Sea in Europe, related species comprises shallows with a sandy or granular large areas become dry during low tide. Crabs retreat into bottom. Most colonies are found encrusting the outside of remaining tidal ponds or protect themselves under bundles gastropod shells inhabited by pagurids (hermit crabs). Col- of wet algae. onies are occasionally found on other substrata, such as the • During stormy and rainy periods and during the winter, shells of living snails and bivalves, piles, or simply stones the crabs retreat into deeper waters. that are exposed to strong tidal currents, so that sand is not • From time to time crabs assemble at common locations, deposited permanently and planktonic food frequently passes lured either by a rich source of food or by sexual partners. with the tidal currents. Young colonies derived from meta- Such aggregations favour not only fertilization of the crab’s morphosing planula larvae (Fig. 5) consist of feeding polyps own eggs but also encounters between the gametes re- connected with each other through a network of gastrovascular leased by symbiotic colonies of Hydractinia.

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

Finding a mobile home through the beating of their cilia, to an elevated location, be How can a planula detect a gastropod shell carried around it a grain of sand or a stone, and adhere to its surface by by a hermit crab, and how can it climb onto the shell? In fact, means of mucus secreted at their anterior, blunt end. Here the planulae of Hydractinia are not able to locate shells occu- they remain standing in a vertical posture until a solid object pied by crabs, nor do they climb onto shells. The spindle- is dragged past. Quick movements of the object, in fits and shaped planula larva of Hydractinia is equipped with neuro- starts, stimulate competent planulae to attach to its surface sensory cells located at or near the anterior pole and also on by discharging the sticky threads of nematocysts (atrichous its tapered posterior tip. Here in the posterior region of the isorhizas and desmonemes) that are found only in the larvae larva, in addition, numerous nematocysts are found (Weis et and are clustered in their tapered posterior region (Weis and al. 1985; Weis and Buss 1986). Buss 1986). Thus the larvae are transferred to the moving This equipment enables the larvae to display a behaviour object, which, hopefully, is a gastropod shell. that enables it to attach to a quickly moving shell (Müller et Discharge of the nematocysts is facilitated by the velocity al. 1976). The planulae are positively phototactic and glide, gradient in the fluid between the larva and the passing sur-

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face. The drag of the moving solid is transmitted to the liq- Fig. 6. Transfer of a planula larva of Hydractinia to a moving uid, so that the liquid layer close to the surface is also set in object such as a gastropod shell carried around by a hermit crab. motion. Thus, a velocity gradient is set up in the shear plane The impact of collision and the velocity gradient of the water surrounding the object (Fig. 6). The change in velocity along around the moving object cause the discharge of the sticky the larva, in addition to the impact of the collision, consti- threads of nematocysts (atrichous isorhiza). tutes 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 re- sume 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 effective only if they are attached to a solid object, not when surfaces of solid objects in the marine environment (e.g., grown in conventional breeding media and presented in a sus- Corpe 1970; Dang and Lovell 2000). These bacteria attach pension. The bacteria do not release a metamorphosis-inducing to the surface of solid objects by means of particular fimbriae substance into the medium; rather, the larvae must come into or pili (species of Pseudomonas) or by secreting sticky cap- close contact with the bacterial layer, and the bacteria are par- sular polysaccharides or other cementing substances. In the ticularly effective if they have been starved for some hours. marine environment, bacteria prefer to attach to solid objects This observation suggests that the metamorphosis-inducing cue because the surface binds and accumulates nutritive mole- is a component of the outer bacterial wall or of the fimbriae cules such as amino acids dissolved in seawater. Bacteria produced by the adhering bacteria. When starved, marine bac- that colonize mollusc shells, in addition, find the organic teria produce specific exopolysaccharides, which confer upon layer of the periostracum, which they may attack and use as the previously free-swimming bacteria the ability to adhere to a source of nutrients. surfaces (in this case the filter membrane) (for example, see In the marine environment, microbiologists interested in Wrangstadh et al. 1990). By applying an osmotic shock, the ecology find bacteria predominantly from the genera Altero- metamorphosis-inducing principle could be released from monas and Pseudoalteromonas on solid surfaces (e.g., Acinas living and surviving bacteria (Müller 1973a, 1973b), but its et al. 1999; Dang and Lovell 2000), and species of these chemical nature has not yet been definitively identified. The genera are among those that have been identified as colo- metamorphosis-inducing principle appears to be a macro- nists of shells occupied by hermit crabs, and as the source of molecule, as it does not pass through ultrafilters with exclusion metamorphosis-inducing activity. In a pioneering study, it limits of about 100 kilodaltons. At present, our working hy- was shown that planulae of H. echinata can be induced to pothesis is focused on hydrophobic proteins or lipopolysac- undergo metamorphosis by presenting them with a film of charides (Bläß 1997). On the other hand, whereas the larvae of such bacteria on filter membranes (Müller 1969). Several the bryozoan genus Bowerbankia can be caused to settle and bacterial species and strains isolated from the micro- metamorphose by providing them with a hydrophobic solid environment of settling planulae were found to be effective, alone (Müller et al. 1976), hydrophobic surfaces per se do not but these amounted to only a small fraction of the strains induce settlement and metamorphosis of Hydractinia planulae. tested with a standard assay protocol (Müller 1969, 1973a, The bacterial inducer is effective not solely by virtue of its 1973b; Wittmann 1977). In subsequent studies, effective physical properties. bacterial strains were identified as Alteromonas haloplanctis Using the initiation of metamorphosis as the criterion, it is and Alteromonas macleodii, as well as some belonging to to be expected that in nature these bacteria are present at the genus Oceanospirillum (W.A. Müller, unpublished data). high density and in effective condition on mollusc shells and Another bacterial species isolated from shells colonized by occasionally on other large solid objects, but not on sand Hydractinia was identified as Alteromonas espejiana (Leitz grains. and Wagner 1993), now called Pseudoalteromonas espejiana. The characteristic vibrating movements of the shell car- Bacteria with metamorphosis-inducing capacity are found on ried by a hermit crab might be another stimulus that induces many substrata (Kroiher and Berking 1999), and frequently metamorphosis, as proposed by Cazaux (1961). However, as belong to the genera Alteromonas or Pseudoalteromonas he was unaware of the significance of the bacteria on the (Acinas et al. 1999; Dang and Lovell 2000). These genera shells, his experiments were not conducted under sterile con- are known to be frequently associated with higher organisms ditions. and to produce biologically active extracellular agents (Holmström and Kjelleberg 1999). The presence of such Artificial metamorphosis inducers and the primary bacteria on solid substrata easily explains the occasional set- mechanism of induction tlement of Hydractinia on substrata other than gastropod Metamorphosis in Hydractinia can be induced artificially shells carried around by hermit crabs. and conveniently with great efficiency by either (i) bathing Interestingly, all bacteria tested so far in our laboratory are the larvae for some hours in seawater enriched with lithium

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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 RT× []K+ U = ln outside cesium ions, Cs+ being the most potent (Müller 1973a; Müller volt × + ZF []K inside and Buchal 1973), or (ii) treating the larvae with activators of protein kinase C (PKC), such as tumour-promoting phorbol This classical equation describes the dependence of the elec- esters or certain diacylglycerols like dioctanoylglycerol (diC8) tric membrane potential on the difference in concentrations (Müller 1985; Leitz and Müller 1987; Leitz 1993; Schneider of cations, here K+, between the exterior and interior of a and Leitz 1994). cell with a semipermeable membrane. For details see text- Subsequent to these findings, many other (less effective) books of neurophysiology (Fig. 7). + + + + + inducers have been found, among them NH4 (Berking 1988a). Cs ,Rb,Li, and likewise NH4 , on the other hand, are Our interpretations of these findings converge in a common known to be transported into excitable cells via the mem- hypothesis about the primary mechanism of induction. We brane-associated ion pump known as Na+,K+-ATPase. This think that all inducing agents act by stimulating excitable pump does not discriminate well between the rare mono- + + + + neurosensory cells such as are found near the anterior pole valent cations Cs ,Rb,Li, and NH4 and its natural-load of the larva (anterior with respect to the direction of its K+. From time to time potassium ions must be pumped into movement when gliding over the substrate). The ions used to the cell to replace those lost by diffusion through the electro- trigger metamorphosis are known to cause depolarization, genic K+-leak channels. Owing to the activity of this pump and thus stimulation, of excitatory cells. Elevated levels of and its low specificity, with time any excitable cell will ac- + + + + + external K directly cause depolarization according to the cumulate Cs ,Rb,Li,orNH4 if they are present. (If Nernst equation: Na+,K+-ATPase is blocked with ouabain, Rb+ and Li+ are

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Fig. 8. Transplantations documenting the transmission of an more, while a highly effective bacterial inducer must be internal metamorphosis-triggering signal (putatively present for merely 20–30 min. neuropeptides) from the anterior part of the larva to the posterior part. (a) Control: a posterior larval fragment grafted onto a non- The supporting influence of mechanical stimuli induced anterior part does not undergo metamorphosis. (b) Only The sensory cells of Cnidaria frequently respond to com- the anterior part was induced (and can be induced) to initiate binations of chemical and mechanical stimuli. The support- metamorphosis by an external stimulus. The grafted posterior ing influence of mechanical shearing forces is indicated by part did not receive an external inducing stimulus; nevertheless it the dependence of dose-response curves not only on the participated in metamorphosis because it received an internal concentration of cesium ions but also on the degree of hydro- stimulus that travelled from the anterior part of the larva to the phobicity of the substrate (Berking and Walter 1994; Berking posterior part. (c) Posterior fragments are not able to respond to 1998). Hydrophobic surfaces have a promoting action and external stimulation by bacteria or cesium ions. (d) Posterior shift the apparent KM of the dose-response curves for cesium fragments respond to neuropeptides of the GLWamide class by to lower values. Hydrophobic adhesion provides shearing metamorphosing. 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).

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 + almost ineffective, but very high doses of Cs still induce remains in the larval state, but when posterior fragments are metamorphosis (Müller 1973a). Driven by high concentra- grafted onto anterior fragments previously induced to metamor- + tion potentials, Cs presumably penetrates the cell mem- phose shortly before transplantation (Fig. 8), the posterior frag- brane also through leak channels.) ments undergo metamorphosis too. They receive a message In the interior of the cells the infiltrating cations cause de- from the anterior region inviting the posterior region of the lar- polarization through the combination of two effects: (1) They val body to participate in metamorphosis (Müller et al. 1976; occupy anionic sites and thus neutralize the surplus of electro- Schwoerer-Böhning et al. 1990). A hypothesis concerning the negative charge that exists inside resting excitable cells. existence of internal signals of neuronal origin has also been (2) They enter and block the electrogenic K+-leak channels proposed, based on histological, cytochemical, and pharmaceu- from inside the cells; blocking these channels prevents re- tical studies in other hydrozoans, e.g., Halycordyle disticha polarization because no positive charge can leave the cell (Kolberg and Martin 1988; Martin and Archer 1997). (Fig. 7). Efforts to isolate and identify the molecules that transmit Depolarization by these indirect mechanisms takes time, this internal signal in Hydractinia culminated in the identifi- so the larvae must be bathed in Cs+ solutions for4hor cation of a novel class of neuropeptides (Leitz et al. 1994b;

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Leitz and Lay 1995; Gajewski et al. 1996, 1998; Leitz 1998, Fig. 9. Planula larva with neurosensory cells labelled with anti- 2000). These share the carboxy terminal consensus sequence bodies against neuropeptides ending with the aa sequence -Gly-Leu-Trp-NH2 (GLWamide) and cause not only anterior GLWamide or RFamide. 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 immunocyto- outer medium or released from internal stores interacts with chemistry with antibodies generated against PPGLWamide (or photoproteins, and this interaction results in emission of light. the GLWamide terminus alone) stained cells with the ex- When the calcium transients were inhibited with calcium chan- pected properties (Fig. 9). That is, they were neurosensory nel blockers, metamorphosis was also blocked. cells located in a belt near the anterior pole with their axonal The calcium transients did not occur during phorbol ester- fibres extending along the mesogloea to the posterior pole induced metamorphosis, indicating that the transients play a (Schmich et al. 1998b). This organization is suggestive. We role upstream of PKC activation. On the other hand, tran- think that these fibres convey the message to the remaining sients can be released with various treatments that do not body parts. The neuropeptides may be released along the induce metamorphosis. Therefore, we think that calcium length of the fibres and (or) at their terminal. transients have a preparatory and supporting function but do Our interpretation does not aim to completely account for not ultimately trigger metamorphosis. all biochemical processes associated with metamorphosis. Berking and co-workers propose triggering mechanisms based Abandoning the juvenile state and the putative role of + taurine and methyl betaines on elevated internal levels of NH4 , the presence of methy- lation potentials, and synthesis of polyamines (Berking 1991, Since nonstimulated Hydractinia larvae remain in the lar- 1998; Berking and Walther 1994). We agree with those au- val state for weeks until they eventually disintegrate, the thors in assuming that entering metamorphosis presupposes presence of substances that have a juvenile hormone-like the release of internal blockades (see below). 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 Putative additional signals synchronizing quantities, is released into the surrounding medium upon in- metamorphosis, such as calcium transients duction of metamorphosis, and prevents larvae from entering Experience with other developing systems suggests that na- metamorphosis if applied externally (Berking 1988b). ture often operates not with one single signalling molecule or Three other metamorphosis-inhibiting compounds isolated signalling system but with several parallel signal-transmission from planulae share the potential to provide methyl groups in systems to ensure a reliable result. The cDNA coding the transmethylation pathways (Berking 1986a, 1986b, 1987). All GLWamide peptides contains one more sequence for a puta- these substances are members of the family of betaine com- tive peptide. This has not yet been synthesized and tested. pounds: N-methylpicolinic acid (homarine), N-methylnicotinic Supporting functions in eliciting particular downstream re- acid (trigonelline), and N-trimethylglycine (glycine betaine). sponses might be fulfilled by other types of molecules, partic- Like taurine, they are present in large quantities (several ularly lysophosphatidylcholine (Leitz and Müller 1991) and millimoles overall concentration in oocytes and larval tis- arachidonic acid and derivatives of this versatile putative sig- sue), and in micromole concentrations they reversibly inhibit nalling substance, collectively known as eicosanoids (Leitz et metamorphosis. During metamorphosis the internal content al. 1993, 1994a). All these substances do not trigger meta- of these betaines declines. Berking (1986a) ascribes the morphosis by themselves, at least not efficiently, but may sup- inhibitory activity of these substances to their potential to port induction by cesium ions. methylate as yet unknown targets. An extension of the hy- In addition to chemical transmitters of messages, another pothesis assigns a second role to these methyl donors during signalling system must be taken into consideration. Using post-metamorphic development. They might control spacing highly sensitive photomultipliers Freeman and Ridgeway of polyps along the stolons of colonial hydroids such as (1987, 1990) detected waves of emitted light travelling along the Eirene viridula (Berking 1986a). body of planula larvae induced to metamorphose by exposure to bacteria or CsCl. The planulae were not Hydractinia but Patterning in the primary polyp Eutonia victoria, Mitrocomella polydidemata, and Phialidium The Cnidaria possess only one axis of asymmetry: the an- gregarium. In contrast to the planulae of Hydractinia, the terior–posterior axis of the planula larva, from which the planulae of these species not only contain an endogenous aboral–oral axis of the polyp is derived. The posterior end of photoprotein but also are translucent. The waves of light are the larva corresponds to the oral pole of the primary polyp. preceded by epithelial action potentials travelling along the The body axis of the future primary polyp has already been surface of the larva. The potentials are probably carried not specified during early oogenesis (Freeman 1981). by sodium ions but by calcium ions, and are propagated In oocytes the nucleus is shifted from the centre of the from cell to cell through gap junctions (like those in vertebrate cell into a peripheral position. It becomes visible through the heart muscle). Calcium entering the epithelial cells from the translucent wall of the gonad (gonophore) as a large “germi-

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nal vesicle”. The site on the egg surface below which the gration. Nematoblasts of the various types enter the ecto- nucleus resides is conventionally designated the animal pole. derm and colonize distinct locations in the larval body. The It is the site of polar-body formation during maturation and distribution pattern may be the result of selective migration the only place where the sperm can enter the egg cell. It is of predetermined cells to their destination (Pennaria tiarella also the site where the first cleavage furrow starts (Fig. 5). and Halocordyle disticha, Martin and Thomas 1980; Martin The animal pole corresponds to the tapered posterior pole of and Archer 1986; Martin 1988a, 1988b, 1992; P. gregarium, the larva, which during metamorphosis gives rise to the oral Thomas et al. 1987; Hydractinia, McFadden et al. 1984; pole of the primary polyp. However, the early specification Weis et al. 1985). of the body axis does not result in early irreversible determi- In Hydractinia, interstitial cells appear to be moving through nation. Centrifugation and cutting experiments revealed a the mesogloea beginning 4 h post insemination and continu- high capacity to regulate (Freeman 1981, 1987, 1990). ing throughout the later stages of metamorphosis. Once in In larvae competent to metamorphose, cell proliferation the ectoderm, the interstitial cells reside at the base of the and cell differentiation are almost at a standstill. They re- ectoderm along the mesogloea, often occurring in clusters of sume when the larvae enter metamorphosis (Plickert and 3 or 4 cells (Van de Vyver 1964; Weis and Buss 1986). Dur- Kroiher 1988; Plickert et al. 1988). These events follow an ing the final stages of metamorphosis, the interstitial cells internal program that is specified during embryogenesis. Al- move from the basal region of the primary polyp into the de- though the planula larva lacks overt longitudinal organization veloping stolons. in its external appearance, the body pattern of the primary polyp is generated during metamorphosis by the determining Apoptosis and extrusion of larval cell types influence of a covert anterior–posterior prepattern that is en- By definition, metamorphosis comprises the decomposition coded in the internal organization of the larva and based in of larval structures and the elaboration of adult structures. The part on stored mRNA (Eiben 1982). When larvae are stimu- planula of Hydractinia is characterized by five ectodermal cell lated to enter metamorphosis and are cut into pieces imme- types (Weis and Buss 1986): (1) epitheliomuscular cells, which diately after induction, the most anterior fragment forms in larvae bear a cilium, (2) gland cells, (3) neurosensory cells, only stolons, whereas the most posterior fragment forms (4) ganglionic nerve cells, and (5) nematocytes. Only during only the head (Fig. 8; Kroiher et al. 1990; Schwoerer-Böhning metamorphosis does the ectoderm acquire interstitial cells from et al. 1990). The development of this prepattern in gastrula- the endoderm. tion and the realization of the final pattern in metamorphosis Larval cells that fulfill transitory functions in presettlement are accessible to experimental interference (Berking 1984, behaviour and metamorphosis are (i) the neurosensory cells 1987) and can be dramatically changed by a low molecular found clustered at the tapered end of the competent planula; weight factor of unknown chemical structure, termed the these probably perceive the impact of collision with a passing proportion-altering factor. This factor also stimulates the for- shell; (ii) the nematocytes, also distributed in the posterior re- mation of nerve cells in developing adults (Plickert 1987, gion and used to anchor the larva onto the shell; (iii) the 1989, 1990; Kroiher and Plickert 1992). The primary signals gland cells, which are concentrated at the anterior end and se- controlling patterning along the anterior–posterior axis in crete a cement at the onset of metamorphosis to permanently larvae and the apex–base axis in polyps are likely to be the fix the larval body to the substrate, and which disappear in same, but the interpretation of these primary signals by the late metamorphosis; (iv) metamorphosis also includes the dis- individual cells changes during metamorphosis (Kroiher 2000). appearance of those ectodermal GLWamide-positive neuro- A hypothetical, hierarchical model of pattern formation sensory cells that are located near the anterior end and were has been put forward by Berking (1998). Patterns of asym- probably the recipients of the external triggering signal (Schmich metrical morphogen distribution provide positional informa- et al. 1998a), as well as the disappearance of RFamide-positive tion. The density of these sources along the body column, or cells (Plickert 1989). A similar reorganization of the nervous their content of stored morphogen precursors, constitutes a system has been described for P. tiarella (Martin 2000). tissue property known in developmental biology as posi- Considerable parts of the larval cellular inventory are re- tional value. With respect to the actual processes occurring moved by apoptosis followed by cell shedding and probably at the cellular level, the distribution pattern of morphogens phagocytosis. Apoptosis can be recognised as early as may merely provide global positional cues to which the vari- 20 min after induction of metamorphosis, before the larvae ous cell types respond very differently according to their reach the point at which development of adult features is ini- previously specified fate. tiated (Seipp et al. 2001).

Exchange of cellular inventory Acquisition of adult characteristics The ectodermal epithelial muscle cells lose their cilia, while Occurrence of interstitial stem cells the endodermal cells acquire a cilium with associated microvilli. Microscopic examination of hydroid embryos, larvae, and The endoderm is completed by two types of gland cells; these metamorphosing specimens has mainly focused on the ori- are concentrated in the hypostome. Unexpectedly, several of gin of the interstitial cells, the founder cells that give rise to the endodermal cells in the young hypostome transitorily contain neurosensory cells, ganglionic nerve cells, nematocytes, and transcripts for the GLW-amide precursor protein and display germ cells (for a review see Thomas and Edwards 1997). In GLWamide immunoreactivity (Schmich et al. 1998a, 1998b; the embryos of the Hydrozoa, interstitial cells generally arise Gajewski et al. 1996). as a central core of cells in the endoderm and eventually The nerve net becomes restructured. While nerve cells are migrate through the mesogloea to the ectoderm. Interstitial absent in the young primary polyp GLWamide-positive, adult cells and nematoblasts undergo extensive long-distance mi- feeding polyps possess many putative nerve cells displaying

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GLWamide precursor transcripts; they are scattered in the ecto- in foamy surf along rocky coasts induces sinking and the on- derm along the body column except in the upper hypostomal set of metamorphosis before actual settlement takes place region (Gajewski et al. 1996). This region is occupied (Kroiher and Berking 1999). by nerve cells exhibiting immunoreactivity against neuropeptides with the carboxy terminus Arg-Phe-amide (RFamide) Anthozoa (Grimmelikhuijzen 1985). From the Anthozoa, too, examples of settlement and meta- At the late stages of metamorphosis, both nematoblasts morphosis in response to bacterial films covering the substrate and nematocytes, complete with cnidocil, begin to move into can be adduced. One of these two representative anthozoans the emerging tentacles. When secondary polyps are formed is the octocoral Heteroxenia fuscescens (Henning et al. 1991, on the elongating stolons, they are colonized by nematocytes 1993) and the other is Acropora willisae. The bacteria were immigrating from the stolonal compartment where their isolated from crustose algae and belong, once more, to the ge- stem cells reside. nus Pseudoalteromonas (Negri et al. 2001). Moreover, induction of metamorphosis by environmental Gene expression bacteria is a phenomenon now known from many sedentary Studies on gene expression in cnidarians in general, and invertebrate species (for a comprehensive but no longer com- metamorphosing Hydractinia in particular, are still fragmen- plete review see Pawlik 1992). tary. Using the methods of reverse genetics, attention was paid to homologues of selector genes known to control de- Physiological mechanisms velopment and patterning in other systems. Remarkably, a gene indicative of “head” in various animal phyla, ems,is Artificial inducers and the mechanism of induction also expressed in the “head” of Hydractinia polyps (Mokady In most species known to respond to bacteria, and also in et al. 1998). The Hox gene Cnox-2 is expressed in the lower species that perhaps respond to other, as yet unknown envi- region of polyps and the stolonal compartment (Cartwright ronmental cues, the effects of natural inducers could be dupli- and Buss 1999). cated in the laboratory by substituting potassium or cesium ions, or activators of PKC (for a review see Leitz 1997). A comparative review of metamorphosis in Only the swimming buds of Cassiopea failed to respond to ionic stimuli with transformation (Müller at al. 1976), but other Cnidaria they responded to treatment with the PKC activators TPA Hydractinia has attained the status of a model organism in and diC8 (Siefker et al. 2000). Besides potassium ions, PKC research on the metamorphosis of marine invertebrates (Had- activators might be the most universal artificial inducers. field 1998). We restrict our review to research on Cnidaria. These experiences reveal a common strategy used by inver- With respect to other organisms we refer to the reviews by tebrate marine larvae to cope with the demands of their life Chia and Bickel (1978), Pawlik (1992), Rodriguez et al. 1993, cycles: all these larvae have to detect a key environmental and Hadfield (1998). The common denominators of compara- stimulus by means of sensory cells, and most sensory cells tive studies are the inductive role of environmental bacteria can be artificially stimulated by elevated levels of external and stimulation by depolarization of sensory cells used to re- potassium or by activators of key enzymes in signal- cognise bacteria-borne or other environmental cues. transduction systems.

Induction of metamorphosis by bacteria Internal signals Of course, not all sensory cells use the same transmitter to Hydrozoa transmit their message to target larval cells. In H. disticha, Following the pioneering study on Hydractinia (Müller but not in Hydractinia, catecholamines trigger metamorpho- 1969), distinct populations of substrate-borne bacteria were sis (Edwards et al. 1987). Fluorophores indicative of the shown to induce metamorphosis in a variety of hydrozoans, presence of catecholamines produced blue–green fluores- including E. victoria, M. polydiademata, P. gregarium (Freeman cence in the anterior region of the planulae (Kolberg and 1981; Thomas et al. 1987, 1997; Freeman and Ridgeway Martin 1988). In P. gregarium serotonin appears to trigger 1990), and H. disticha (Edwards et al. 1987). and synchronize the internal processes of metamorphosis (McCauley 1997). Although serotonin is not among the induc- Scyphozoa ers of metamorphosis in Hydractinia, a serotonergic mecha- Planulae and propagules of some Scyphozoa have also nism might be involved in downstream secondary processes, been successfully induced to enter metamorphosis using bac- since an inhibitor of serotonin synthesis retarded metamor- teria found in the environment of settled polyps. In particular, phosis (Walther et al. 1996). larvae of Cassiopea andromeda responded to a species of Vibrio alginolyticus (Neumann, 1979; Hofmann et al. 1984, Release from inhibition 1996; Fitt and Hofmann 1985; Fitt et al. 1987; Hofmann and In the swimming buds of Cassiopea a particular mecha- Brand 1987), the propagules (pedal stolons) of Aurelia aurita nism that controls development is operating. The buds enter to a species of Micrococcaceae (Schmahl 1985a, 1985b), and metamorphosis without being stimulated by external cues planulae of Cyanea capillata and A. aurita were reported to when their anterior part, which in normal development gives settle on solid objects covered with a film of (unidentified) rise to the basal disk and stalk of the scyphistoma (polyp), is bacteria (Brewer 1976, 1978, 1984; Schmahl 1985a, 1985b). removed. The posterior fragment will differentiate into a However, A. aurita does not respond to only one external free-swimming scyphistoma (Müller et al. 1976). The ante- stimulus. The water–air interface of the air bubbles that occur rior portion of the body is apparently the source of activity

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Fig. 10. A stolon tip contacting the flank of an established sto- • Gregarious settlement of non-isogeneic individuals favours lon in Hydractinia. Nematocytes of a special type (microbasic exchange of alleles and rapid distribution of those alleles mastigophores) migrate to and assemble at the contact site. In that are favourable under the particular local environmen- encounters between histoincompatible colonies the nematocytes tal conditions. are discharged in order to kill and eliminate the competitor for Disadvantages include the following: living space. • Predators conveniently find a richly laid table, and infec- tions spread readily through the community. • Aggregated adults must compete for food, which may re- duce 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 that inhibits further development. Application of cholera toxin species living on specific, spatially limited substrata will (Wolk et al. 1985), known to interfere with G protein-coupled sooner or later come into conflict with allogeneic neigh- signal transduction, ammonium ions (Berking and Schüle bours and must be eager to eliminate such competitors. 1987), and the protein phosphatase inhibitor cantharidin (Kehls This is the case in Hydractinia, therefore we return to this et al. 1999) mimics the removal of this source of inhibition. model organism once more, while not forgetting parallel phenomena in other genera. Benefits and costs of gregarious settlement: mutual support versus civil war Allorecognition responses and elimination of competitors Various taxa of sedentary marine invertebrates, including Advantages versus disadvantages of gregarious settlement cnidarians such as sea anemones, colonial corals, and colo- Benthic sedentary invertebrates such as barnacles, mussels, nial Hydrozoa, are known to possess a complex array of echiurids, sabellariid polychaetes, and ascidians are most responses, primed to nonself tissue contacts (reviewed by frequently found in dense clusters. Most cnidarians also pre- Grosberg 1988). Members of cnidarian populations are able fer to live with conspecifics. In fact, the largest community to discriminate nonself from self and can efficiently react of related animal species, and the largest residential pre- against various allogeneic and xenogeneic challenges. Sea mises ever constructed by living organisms, is the Great Bar- anemones, which are able to change their position, are rier Reef along the eastern coast of Australia. Although large caused to leave their place of residence by intolerant neigh- reefs are composed of many species, the distribution of indi- bours when the latter discharge nematocysts mounted on vidual members and their kinship in the reef community are knob-like protrusions of the body wall, called acrorhagi, or not random. Colonies composed of individuals that are ge- sling nematocysts-bearing filamentous acontia out of their netically related or identical (clones) are formed by asexual mouths or through openings in the body wall towards un- division of an initial settler, as in the growth of corals, or by wanted competitors for living space. Corals may try to harm the settlement of short-term larvae near their mothers. Ag- and weaken their allogeneic neighbours during toxic interac- gregations of genetically different individuals may also be tions and to overgrow them (Frank and Rinkevich 1994; formed by the settlement of planktonic larvae on or near Rinkevich et al. 1994; Frank et al. 1995, 1997). However, all adult conspecifics. This last condition is particularly preva- these interactions occur long after metamorphosis has taken lent among hard-bottom, sessile intertidal organisms, includ- place. ing sea anemones and hydrozoans. Epibiotic invertebrates In Hydractinia, the ability to discriminate between self and may become aggregated as a result of larval preferences for nonself matures during metamorphosis, and defence against settling on particular living substrata. allogeneic competitors is possible as soon as metamorphosis Gregarious settlement has several advantages, but is not is completed (Fuchs et al. 2002). When two primary polyps without costs. There are clear trade-offs between the advan- or colonies come into contact by way of their extending sto- tages and disadvantages, but the prevalence of gregarious- lons, either the stolons fuse, in which case a chimæra results, ness in hard-substrate marine communities suggests that the or they do not fuse, in which case one eliminates the neigh- benefits outweigh the costs. The benefits are manifold: bouring competitor for living space. The competitor is at- • When larvae settle on or near adult conspecifics, they tacked by means of nematocytes of a particular type, classified choose a habitat that is more likely to support postlarval as microbasic mastigophores. These are only found in the growth than if they had settled indiscriminately. stolonal compartment of the colony, patrolling permanently • Proximity increases the chances of successful fertilization along the stolons. The nematocytes accumulate at contact sites for both internally fertilizing and freely spawning species. (Fig. 10), direct the cnidocils toward the neighbour, and sud- Synchronization of spawning can be improved by the re- denly discharge their poisons into the foreign tissue. Upon release of pheromones. peated and mutual attacks, one of the two competitors loses

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and becomes paralyzed (Müller 1964; Buss et al. 1984; Lange Berking, S. 1988b. Taurine found to stabilize the larval state is re- et al. 1989; Buss 1990; Buss and Shenk 1990; Lange et al. leased upon induction of metamorphosis in the hydrozoon 1992). Hydractinia. Roux’s Arch. Dev. Biol. 197: 321–327. Incompatibility and attacks are always observed in colo- Berking, S. 1991. Control of metamorphosis and pattern formation nies derived from different parents but less frequently among in Hydractinia (Hydrozoa, Cnidaria). BioEssays, 13: 323–329. close kin (and never when the two colonies belong to the Berking, S. 1998. Hydrozoa metamorphosis and pattern formation. same clone, a situation that occurs only in the laboratory). Curr. Top. Dev. Biol. 38: 81–131. Based on the outcome of crosses, it has been proposed that Berking, S., and Schüle, T. 1987. Ammonia induces metamorpho- allorecognition is genetically controlled by a single, yet highly sis of the oral half of buds into polyp heads in the scyphozoan polymorphic genetic locus with codominantly expressed al- Cassiopea. Roux’s Arch. Dev. Biol. 196: 388–390. leles. Sharing at least one allele at this locus enables two Berking, S., and Walther, M. 1994. Control of metamorphosis in colonies to fuse, whereas rejection results when no allele is the hydroid Hydractinia. In Perspectives in Comparative Endo- shared (Hauenschild 1954, 1955; Grosberg et al. 1996; crinology: Invited Papers from the XII International Congress of Mokady and Buss 1996, 1997). Sharing an identical allele is Comparative Endocrinology, Toronto, Ont., 16–21 May 1993. Edited by K.G. Davey, R.E. Peter, and S.S. Tobe. National Re- unlikely in distantly related conspecifics. search Council of Canada, Ottawa, Ont. pp. 381–388. The phenomenon has received two different interpretations, Bläß, O. 1997 Metamorphoseauslösung bei Hydractinia echinata. which, however, are not contradictory but complementary. Wissenschaftliche Arbeit vorgelegt der Fakultät für Biologie, (1) Nonfusion prevents the invasion of foreign primordial Universität Heidelberg, Heidelberg, Germany. germ cells, which in fact can colonize the neighbouring tis- Brewer, R.H. 1976. Larval settling behavior in Cyanea capillata sue and displace the germ cells in the gonads (Müller 1964, (Cnidaria, Scyphozoa). Biol. Bull. (Woods Hole, Mass.), 150: 1967). This is tolerable only when the two colonies are sib- 183–199. lings and therefore share many alleles at other genetic loci Brewer, R.H. 1978. Larval settlement behavior in the jellyfish Aurelia also. The reduction of fitness in one partner might be com- aurita. Estuaries, 1: 120–122. pensated for by avoiding the costs of war and through the Brewer, R.H. 1984. The influence of the orientation, roughness, and benefits of fusion. Upon fusion, the enlarged common col- wettability of solid surfaces on the behavior and attachment of ony can occupy the valuable substrate faster than a single planulae of Cyanea (Cnidaria: Scyphozoa). Biol. Bull. (Woods colony could do. (2) If, however, the two competitors are Hole, Mass.), 166: 11–21. allogeneic and share only a few alleles, each colony must Brooks, W.R., and Gwaltney, C.L. 1993. Protection of symbiotic take care to ensure its own fitness (Yund et al. 1987; Hart and cnidarians by their hermit crab hosts: evidence for mutualism. Grosberg 1999). Because hard substrata in general, and shells Symbiosis, 15: 1–13. inhabited by hermit crabs in particular, are very rare in the Buss, L.W. 1990. Competition within and between encrusting clonal habitat of Hydractinia, an allogeneic competitor is better invertebrates. Trends Ecol. Evol. 5: 352–356. eliminated than tolerated. Only in H. symbiolongicarpus can Buss, L.W., and Shenk, M.A. 1990. Hydroid allorecognition regu- incompatible allogeneic colonies coexist, in that they deposit lates competition at both the level of the colony and the level of noncellular material to form a barrier between them (Buss et al. the cell lineage. In Defense molecules. Edited by J.J. Marchalonis 1984). Hence, the question arises as to whether competition and C. Reinish. Alan R. Liss, New York. pp 85–106. is less severe in North American habitats than in European Buss, L.W., and Yund, P.O. 1989. A sibling species group of habitats. Thus, we return to ecological aspects: metamorpho- Hydractinia in the north-eastern United States. J. Mar. Biol. sis in cnidarians can be understood only when these are Assoc. U.K. 69: 857–874. taken into account. Buss, L.W., McFadden, C., and Greene, D.R. 1984. Biology of hydractiniid hydroids. 2. Histocompatibility effector system/ competitive mechanism mediated by nematocyst discharge. Biol. 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