The Gastropod Nervous System in Metamorphosis Rene Marois' and Thomas J. Carew2

'

Program in Neuroscience and 2Departrnents of Psychology and Biology, Yale University, New Haven, Connecticut 06520

SUMMARY Many gastropods, including the sea hare Apiysia culifornicu, undergo metamorphosis in passing from the larval to the juvenile phases of their life cycle. During metamorphosis, the gastropod nervous system is affected by both progressive and regressive neurunal events, In addition to this metamorphic reorganization,

the nervous system appears to be centrally involved in initiating metamorphosis. We propose that gastropods not only possess temporally distinct neuronal adaptations for the specific needs of the larval and juvenile phases, but also another transient neuronal adaptation specialized to subserve the metamorphic episode.

INTRODUCTION

Aplysia ( Kriegstein, 1977a,b; Rankin, Stopfer, Marcus, and Carew. 1987; Nolen, Marcus, and Carew, 1987: Cash and Carew, 1989). Such an approach has revealed some interesting features of gastropod neurogenesis that appear to be uncommon relative to most other invertebrate and vertebrate species, such as the long temporal extent of neurogenesis and the apparent lack, or scarcity, of occurrence of nerve cell death (McAllister, Scheller, Kandel, and Axel, 1983; Jacob, 1984; Cash and Carew, 1989; Moffett and Ridgway, 1988; Croll and Chiasson, 1989). The complete development of the nervous system of species of gastropods that undergo major structural reorganization at metamorphosis may appear to be less tractable for understanding the rules that govern the development of the adult phenotype. However, if one is interested in how the nervous system serves the developing animal, then species with complex life cycles are particularly interesting. For instance, the nervous system of a gastropod larva would be expected to be fundamentally different from that of the adult nervous system, as the different ecological niches occupied by the larva and the adult would appear to necessitate morphological, physiological, and behavioral adaptations unique to each stage. These different demands should be reflected in different neural designs; thus a reorganization ( metamorphosis) of the nervous system appears necessary

A fundamental goal in developmental neuroscience is to achieve an understanding of the rules and principles that govern the formation of the adult nervous system. Progress towards this goal is greatly aided by preparations that show a relatively slow and straightforward developmental pattern such that individual elements of the nervous system, as well as alterations of these elements, can be easily distinguished and followed. Although this approach has long been used in a variety of animals, it has only recently been applied to the gastropods. Since their nervous systems are composed of relatively few, large, and sometimes uniquely identifiable cells, gastropods appear particularly well suited for such study. A developmental analysis can be applied to species of gastropods that either have a direct development, such as in the pulmonate Lyrnnaea (Marois, Chiasson, and Croll, 1987; Croll and Chiasson, 1989) or to a portion of a species' complex life cycle that shows a Straightforward developmental pattern, such as the juvenile development of the opisthobranch

Received June 13, 1990; accepted June 21, lY90 Journal of Neurobiology, Vol. 21, No. 7, pp. 1053-1071 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/07 1053- 19$04.00

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for effecting the transition from larval to adult forms. This requirement is not unique to gastropods; it is common to all metamorphic animals, such as holometabolous insects and amphibians (Fox, 1984: Truman, Levine, and Weeks. 1985; Levine and Weeks, 1989: Weeks and Levine, 1990a,b). In addition to the larval and adult organization of the nervous system, some animals may require the transient existence of a neural Organization unique to the metamorphic period. Although the involvement of the nervous system in the metamorphic process may be ubiquitous to metamorphosing animals, it may have a strikingly distinct role in marine molluscs, such as gastropods. Mctamorphosis in the latter species, unlike that in holometabolous insects and amphibians, typically involves an active sensory process during which the competent larvae must search for, recognize, and finally settle on a highly specific substrate (usually a plant, animal, or microbial organism associated with the cnvironment of the adult) (Hadfield, 1978; Chia and Rice, 1978). Thus the premetamorphic animal must have the appropriate sensory transduction process necessary to respond to particular external metamorphic triggers (Hadfield, 1978). In addition to these sensory events, a critical involvement of the nervous system in gastropod metamorphosis is further suggested by the specific series of behavioral acts that the animal performs during the metamorphic process itself A major limitation in our current knowledge of gastropod neurometamoqhosis is the scarcity of studies performed in this field. This is surprising given the fact that gastropods have becn longtime favorite preparations of both embryologists and neurobiologists. A major impediment in using these animals for developmental studies has been the complex life cycle of many gastropods, which posed significant challenges for raising them in the laboratory. The principal difficulty arises from the fact that the development of gastropods, and molluscs in general, is essentially indirect; the adult body plan does not develop directly from the egg, but undergoes one or more larval forms en route to the final adult phenotype (Raven, 1966; Bonar, 1978a; Fioroni, 1982). Typically, the first ciliated larval form developing from the gastrula is called the truchophorc. In most molluscs, and in all gastropods, the trochophore is then transformed to a veliger, a larval form with a complex pattern of organogenesis. Metamorphosis is necessary for the transition from the larval body structure of the veliger to that of a juvenile. The extent of the metamorphic pro-

cess varies greatly according to the contrast in ecological niches occupied during the larval and juvenile phases of the life cycle of the animal. It is most pronounced in marine molluscs, such as the opisthobranch gastropods, that switch from a freeswimming, pelagic larval phase to a crawling, benthic adult life-style. It is less pronounced but still present in gastropod species with a short freeswimming larval life, or with those that have no free-swimming larval stage and pass through ( a reduced) metamorphosis while still in the egg capsule, hatching as young juveniles. The ease with which the latter species can be raised in the laboratory encouraged much of the early work on them (Thompson, 1958, 1962. 1967; Smith, 1967: Tardy, 1970, 1974; Bonar and Hadfield, 1974; Bridges, 1975; Bonar, 1976, 1978a,b; but see Thiriot-Quikvreux, 1970). Although important insights into development have been gained from species with reduced larval and metamorphic phases, these gastropods allow only a limited assessment of the metamorphic process on species with pronounced free-living larval stages. In addition, except for Tardy’s studies (1970, 1974) on the n udibranch Aeulidrdlu, little information has been acquired about the fate of the nervous system during molluscan metamorphosis, yet this is exactly a developmental period during which one would expect to sec a drastic neural reorganization. Recent successful laboratory rearing of numerous gastropods with complex life cycles. and especially opisthobranch species with free-swimming larval stages, has paved the way for studying the neural changes associated with metamorphosis (Kriegstein, Castelluci, and Kandel, 1974: Switzer-Dunlap and Hadfield, 1977; Kempf and Willows, 1977; Bickell and Kempf, 1983). Even though the developmental pattern of any given molluscan species may depend more on environmental factors than on phyletic relationship (Fioroni, 1982). it is generally agreed that the members of the gastropod class, and more specifically the opisthobranch subclass (Table 1 ), typically show profound metamorphosis (Bonar, 1978a: Hadfield, 1978). Most opisthobranchs are marine, benthic animals. Among the more than 1100 species of opisthobranchs, an extensive divergence of body plans can be found (Hyman, 1967). Nonetheless, the larval forms of most of these species are strikingly similar (Bonar, 1978a). It is adapted for a free-swimming,pelagic life-style, with the variation of either a planktonic (filterfeeder) or lecithotrophic (internal yolk reserves) nutritional mode. Some opisthobranch species do not have a free-swimminglarval stage and hatch as

Gastropod Mrtainorphosis

small adult-like juveniles, but even in thcse cases one can distinguish a more or less atrophied larval form inside the egg capsule (Raven, 1966; Bonar, 1978a; Fioroni, 1982). In addition, most differences in larval morphology can be accounted for by the variations in the requirements for the subsequent juvenile phase (Bickell and Chia, 1979). Thus from a relatively common opisthobranch larval structure must arise, at and around the time of metamorphosis, a number of processes that give rise to the diversity of adult forms. It is within the opisthobranch subclass that most larval and metamorphic studies have been carried out, especially on species of the nudibranch order. However, the nervous system has been extensively studicd in another order, the Anaspidea, both during adulthood (Kandel, 1979) and during development (Kriegstein et al., 1974, Kriegstein, 1977a,b; Bridges, 1975; Schacher, Kandel, and Woolley 1979a,b; Cash and Carew, 1989). As our primary aim is to discuss the gastropod nervous system in metamorphosis, we have focused our review primarily on the anaspid A p l ~ ~ and r a a few nudibranch species as models of gastropod metamorphosis (Table l ). The extent of the metamorphic process varies enormously between species of the more ancestral gastropod subclass, the Prosobranchia, although it is generally more subdued than in opisthobranchs (Fretter, 1969; Fioroni, 1982). Thus we will only refer to selected species of prosobranchs that show pronounced metamorphosis. Because bivalve development has been studied in considerable detail, where appropriate, we will compare the metamorphic patterns of this

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class with those of the gastropods. The vast majority of species of pulmonates, representing the third gastropod subclass, undergo direct development with greatly atrophied veliger and metamorphic stages (Raven, 1975). and the few pulmonate species that do have a free-swimming larval stage undergo a developmental pattern resembling that of opisthobranchs (Little, Stirling, Pilkington, and Pilkington, 1985). For these reasons, pulmonates will not be discussed here. Also excluded from this review will be other molluscan classes that are poorly understood, as well as the Cephalopods, which undergo a drastically different developmental pattern (Raven, 1966; Verdonk, van den Biggelaar, and Tampa, 1983). In this review. we will first discuss the organization of the nervous system in the larval stage of development and then consider how it is modified by the metamorphic process. Next, we examine how thc nervous system itself may be involved in the transduction of the metamorphic signal. Finally, because the organization and function of the nervous system is better understood when analyzed in a functional context, for each of the developmental stages we will also consider the sensory and motor structures that contribute to the general behavioral organization of the animal. LARVAL PERIOD

General Morphology The larval phase in the life cycle of numerous opisthobranchs and other gastropods with free-

Table 1 Taxonomy of the Gastropod Class'

Phylum

Class

Subclass

,--

Order

Phyllaplysia Anaspidea

Prosobranchia Opisthobranchia

Genus

Aplysia Bursatella

Notaspidea, Cephalopoda and others

Aeolidiella Tritonia

Ccphalopoda and other Nudibranchia

Phestilla

Melibe Onchidoris

' Only taxonomic groups relevant to this review are illustrated. (Modified from Kandel, 1Y79)

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,I4arorb and Carew APICAL SENSORY ORGAN

LARVAL

PROPODIUM LARVAL RETRACTOR MUSCLE

Figure 1 Diagrammatic representation of a free-swimming apisthobranch veliger. Only structural features relevant to this review are illustrated in this oblique view. The large larval retractor muscle sends ramifications into the velum and foot of the animal. The indicated position of the apical sensory organ is only approximate. This basic larval design also applies to other gastropod species which have a free-swimming veliger stage.

swimming veligers begins with the hatching of the embryo and ends with metamorphosis. The early portion of the larval phase is marked by physical growth up to a species-specificshell size; however, the veliger is not yet competent to metamorphose, as a period of internal growth and transformation must first occur ( Kriegstein, 197721; Switzer-Dunlap and Hadfield, 1977; Kempf and Willows, 1977; Bickell and Kempf, 1983; Paige, 1988). A typical veliger competent to metamorphose is illustrated in Figure l . It is characterized posteriorly by a globular shell and visceral mass, and anteriorly by the cephalopedal mass. This latter mass is composed of two general structures, the velum and the foot. The velum, which consists of hypertrophied lobular outgrowths from the dorsolateral surface of the head, is the larval organ of both locomotion and feeding; it works by creating water currents with the rapid beat of cilia located along its rim. The other part of the cephalopedal mass is the developing foot. The propodium, the anterior portion of the foot essential for crawling, is acquired only late in larval life. The animal also possesses a larval heart, which appears at midlarval

stage (Kriegstein, 1977a; Switzer-Dunlap and Hadfield, 1977; Paige, 1988). Muscular and Sensory Systems

Bonar ( 1976, 1978a) recognized two muscular systems in opisthobranch larvae. The first is composed of the larval retractor muscles, the size and number (0 to 2 ) of which vary between species (Thompson, 1958, 1967; Smith, 1967; Tardy, 1970; Bonar and Hadfield, 1974; Bickell and Chia, 1979; Bickell and Kempf, 1983). In the two aplysiids Phylluplysiu and Aplysia, there is a large left retractor muscle and a small right one (Saunders and Poole, 1910; Bridges, 1975). These muscles are attached posteriorly on the inner surface of the shell, but anteriorly, they fan out in the foot and the velum (Fig. 1). They are responsible for withdrawing the head and foot into the shell. A separate accessory pedal retractor muscle has also been observed in some opisthobranchs (Bickell and Chia, 1979; Bickell and Kempf, 1983). The second muscular system is a diffuse series of unicellular muscle cells located under the epithelium of the

Gastropod Metamorphosis

cephalopedal mass (Bonar, 197th). A good example of this system is the intricate muscular system of the velum (Carter, 1926; Mackie, Singla, and Thiriot-QuiCvreux, 1976). In addition to these two muscular systems, a third, the mantle and perivisceral retractor muscles, has been recognized in some nudibranchs (Bickell and Chia, 1979;Bickell and Kempf, 1983), and probably occurs in the other opisthobranchs as well. Prosobranchs with a pronounced larval form have a comparable general muscular system (Fretter, 1967, 1972). Bivalves, on the other hand, have a different muscle system; it is mainly comprised of a set of velar retractors, a set of pedal retractors. and a set of adductor muscles (D’Asaro, 1967; Bayne, I97 1; Hickman and Gruffydd, 1971 ) . There are a few major sensory organs in gastropod larvae. First, there is a pair of eyes acquired at midlarval stage (Raven, 1966; Kriegstein, 1977a; Swit7er-Dunlap and Hadfield, 1977; Paige, 1988). Second, there are the statocysts, the sensory organs involved in balance and righting reflexes. They have an embryonic origin (Saunders and Poole, 1910; Raven, 1966), except in bivalves, where they form only late in larval life (Moor, 1983). Only Smith ( 1967) has reported the presence of an osphradium-a chemosensory structure-in premetamorphic larvae of the opisthobranch Retusa ohtusa, although both Smith (1967) and Kriegstein (1977a,b) described the appearance of the osphradial ganglion in veligers of Retusa and Aplysia, respectively. Finally, an unpaired apical, or cephalic, sensory organ has been observed in a central position on the head, between the velar lobes at the level of the shell aperture of many opisthobranchs and other gastropods, as well as bivalves (Fig. 1 ) (D’Asaro, 1967; Tardy, 1970; Bayne, 1971; Hickman and Gruffydd, 1971; Bonar 1978a,b; Morse, Duncan, Hooker, Baloun, and Young, 1980). Based on its ultrastructure. this organ is presumed to be of a chemosensory nature and may be involved in larval settlement (see below ) .

Nervous System In the opisthobranch Apfysia, small paired cerebral and pedal ganglia are already present by hatching (Saunders and Poole. 1910). The paired pleural, and the small paired optic and unpaired osphradial ganglia are distinguishable by midlarval stage. The unfused abdominal ganglia also arise during larval life, although the anlage o f these ganglia are observed in hatchlings (Schacher et al.,

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1979a). The last other pair of central ganglia, the buccal ganglia, emerges just prior to metamorphosis (Kriegstein, 1977a.b). By then the different central ganglia are already linked by commissures and connectives (Kriegstein, 1977b), which probably arose from primary outgrowths of the ganglia ( Raven, 1966). A similar developmental scheme occurs for other opisthobranchs: the cerebral ganglia have an embryonic origin, whereas the rest of the major central ganglia, including the pedal ganglia, arise and grow during the larval period. except for the buccal ganglia, which have a late larval origin (Smith, 1967; Tardy, 1970, 1974; Bickell and Chia. 1979; Bickell and Kempf, 1983; Kempf, Masinovsky and Willows, 1987). However. there are major differences in the developmental pattern. number. and fate of the ganglia of the visceral loop. Although the central nervous system (CNS) of adult nudibranchs does not have ganglia associated with the visceral loop (Schmekel. 1985). such visceral ganglia have been observed in the larvae of some nudibranch species (Tardy, 1970, 1974; Kempf et al., 1987 j . In the nudibranch Arolidiella at least, the larval visceral ganglia were shown to fuse subsequently with the cerebropleural ganglia (Tardy, 1 970, 1974; see below). In other nudibranch larvae, the visceral ganglia have never been observed, either because they are fused from the onset with the cerebropleural ganglia or because they never form (Thompson, 1958, 1962; Bickell and Chia, 1979: Bickell and Kempf, 1983). The formation of the nervous system in prosobranchs resembles that of opisthobranchs (Guyomarc’h-Cousin, 1974; Demian and Yousif, 1975), and even in bivalves all the major central ganglia are layed down dunng the larval period ( D’Asaro. 1967; Bayne, 1971; Hickman and Gruffydd. 1971). All the ganglia arise primarily by cell proliferation from specific, laminated ectodermal zones of the body wall and by subsequent inward migration to form cellular ganglionic masses (Smith, 1967; Tardy, 1970, 1974; McAllister et al., 1983; Jacob, 1984j. Invagination o f ectodermal zones may also contribute to the formation of the CNS (Thompson. 1962; Tardy. 1970, 1974). Behavior

Only a few studies have examined the behavioral role of the larval nervous system (Moor, 1983). It is known that the cerebral ganglia innervate the statocysts (Raven, 1966: Moor, 1983). These gan-

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Marois and (‘arew

glia also send radiating projections to the velum, where a neuritic net appears to keep the coordination of the ciliary beat and velar motion under neural control (Carter, 1926; Mackie et al., 1976; Kempf et al., 1987; Arkett, Mackie, and Singla. 1987). The frequency of the velar ciliary beat may in fact be under serotonergic control as bath application of this neurotransmitter modulates the beat frequency (Koshtoyants, Buznikov, and Manukhin, I96 I ; Gosselin, 196 1 ) and cerebral ganglion cells that send projections into the velum are immunoreactive for serotonin (Marois and Carew, 1989, in preparation). In culture, the gastropod larva typically exhibits a cyclical rising and sinking behavior. It swims upward, being positively geotropic, stops its ciliary beat, sinks, and starts swimming upward again ( Switzer-Dunlap, 1978; Morse, 1985; Paige, 1988). The ciliary beat appears to stop spontaneously, or can be induced to stop by gently touching the velum (Thompson, 1967; Mackie et al., 1976; R. Marois, unpublished observations). This also often results in the withdrawal of the head. velum, and foot inside the shell, with the aperture closed by the operculum. This withdrawal reflex is very rapid and is mediated by the contraction of the large larval retractor muscles. Given the speed and coordination of the withdrawal reflex, it is likely that the larval retractor muscles are neuronally innervated. Whether the diffuse muscular system in the cephalopedal mass is also under neural control is unknown, although there are widespread serotonergic neural projections to the peripheral tissue of Apijairr (Marois and Carew, in preparation). In the late larval stage, the propodium of the foot has developed and is already highly motile. enabling the animal to crawl on the substrate, although the veliger spends most of its time swimming (Kriegstein et al., 1974, 1977a; Switzer-Dunlap and Hadfield, 1977). The visual behavior of the opisthobranch veliger is also poorly understood. There is some innervation of the eyes from the CNS, via the optic ganglia (Tardy, 1974: Kriegstein, 1977a,b; Bickell and Chia. 1979; Kempfet al., 1987). Adult shelled gastropods display a shadow withdrawal reflex, in which the animal withdraws into its shell in response to a sudden decrease in light intensity (Audesirk and Audesirk, 1985). We know of no systematic developmental study of this reflex, although Smith ( 1967) has reported that veligers of the cephalaspid Retusa in their late larval stage withdraw into their shell when exposed to strong illumination.

Young veligers of the nudibranch Phestilla appear to be positively phototactic, being more attracted to the illuminated side of their tanks (Bonar and Hadfield, 1974). However, as metamorphosis approaches, these same veligers avoid the light. Bonar and Hadfield (1974) interpreted this behavioral reversal as a preparatory shift for the veliger to adapt from a pelagic to a benthic life-style, enabling the animal to find the appropriate benthic substrate on which to metamorphose. In contrast, Switzer-Dunlap and Hadfield (1977) did not observe any phototactic behavior in their study of four species of Hawaiian aplysiid larvae. On the other hand, Fretter (1969) argues that many prosobranch veligers are positively phototactic. In addition to possible species differences, the variable phototactic behavior observed in these studies is probably a result of different experimental procedures employed and the age of the larvae at testing. In addition to the behavioral processes discussed above, more complex behaviors have also been observed in some gastropod species. For example, the larvae of the carnivorous nudibranchs Tritonia and Phestilla display an interesting avoidance behavior towards animals that they will actively prey upon after metamorphosis (Harris, 1975; Kempf and Willows, 1977). In the case of Phestilla, it has been suggested that this avoidance behavior results from the fact that these larvae are themselves subject to predation by the very same animals that Phestilla will feed upon following metamorphosis (Harris, 1975). METAMORPHOSIS

Metamorphosis in free-swimming molluscan larvae is closely linked to and preceded by settlement. a behavioral process during which the larva, in response to an inducing signal, ceases swimming, crawls along a substrate, and may then attach to it (Fretter, 1972; Kriegstein et al., 1974, 1977a; Bonar. 1978a; Coon, Bonar, and Weiner, 1985; Chia and Koss, 1988; see below). This process is reversible; if disturbed the animal may cease progressing through the rest of metamorphosis. Settlement is followed by metamorphosis per se, an irreversible morphogenetic rehponse. which involbes the loss ofthe velum and other larval organs and the genesis of adult organs and functions (Fretter, 1972; Kriegstein, I977a; Bonar, 1978a; Coon, Bonar, and Weiner, 1985; Chia and Koss, 1988).

Gastropod Metamorphosis

Behavior during Metamorphosis

Metamorphosis occurs in a relatively short time, 2-3 days for aplysiids (Kricgstein et al., 1974, 1977a; Switzer-Dunlap and Hadfield, 1977. Paige, 1988). Upon presentation of the seaweed Laurencia pucifica, competent veligers of Aplvsiu californica will soon crawl on the algae. The larva may alternate between short swimming and crawling bouts, but it typically stops crawling and. within an hour. adheres to the substrate by means of secretions from the metapodial glands, and then ceases ciliary feeding (Kriegstein, 1977a). The animal retracts almost completely into its shell, except for the planar section of the foot that remains attached to the substrate. The animal at this point is said to have settled, and it remains in this upright position throughout the early stages of metamorphosis [Fig. 2 ( A ) ] . In opisthobranchs that discard their shells (such as in the nudibranchs), this position ensures that the larval retractor muscles have an anchorage against which they can exert the contractile force necessary for their detachment from the shell (Bonar, 1976. 1978a; Bonar and Hadfield, 1974; Bickell and Kempf, 1983). The aplysiids also show this characteristic metamorphic posture. Whether it has any function in these spe-

cies is unclear as the aplysiids do not discard their shells as nudibranchs do, and it is not known if they severe the larval retractor muscle-shell attachment. In fact, juveniles of the closely related Phjdlaplysia have body muscles attached to the shell, although the relationship of this musculature to the larval one is uncertain (Bridges, 1975; see below). Kriegstein and colleagues ( 1974) have closely examined the behavior of Aplysia californica through metamorphosis. The locomotor pattern develops gradually. Crawling, which emerges in the late planktonic phase, first depends primarily on the action of pedal cilia (Bickell and Kempf, 1983). Although the animal is fixed during the early phase of metamorphosis. it can be seen crawling around on the substrate during the late phase [Fig. 2 ( B ) ] . The muscular pedal waves characteristic of the adult locomotory pattern develop mostly during the juvenile period, replacing the ciliary action (Stopfer, Schuerman, and Carew, 1987). Of all behaviors in the animal’s repertoire, it may bc that feeding is the most drastically reorganized at metamorphosis. T h e animal must switch from a planktotrophic or lecithotrophic regime to a herbivorous or carnivorous diet. The

B

A

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FOOT,

-

ANTERIOR TENTACLES

L

Figure 2 Major changes associated with metamorphosis of the free-swimming opisthobranch veliger. ( A ) Early phase of metamorphosis. The veliger, standing in an upright position, is almost completely withdrawn into the shell except for the posterior part of the foot, which is attached to the substrate. h’udibranchs use this posture to pull loose their muscle-shell attachment. The retracted velum has not been discarded yet. ( B ) Late phase of metamorphosis. The animal is now free to crawl on the substrate. The anterior tentacles (for aplysiids) or oral veil (for some nudibranchs) may develop in lieu of the atrophied velum. Unlike anaspids, nudibranchs have discarded their shell by this late phase of metamorphosis.

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extensive reorganiLation of the feeding system nccessitates that the underlying adult organs develop during the larval period if the animal is to feed soon after metamorphosis. In fact, the onset of adult feeding behavior is often considered as the end point of metamorphosis (Thompson, 1958, 1962; Fretter, 1969, 1972; Thiriot-QuiCvreux, 1970; Kriegstein et al., 1974, 1977a; Bridges, 1975; Switzer-Dunlap and Hadfield, 1977; Bonar, 1978a; Bickell and Chia, 1979; Bickell and Kempf. 1983; Paige, 1988; but see Kempf and Willows, 1977). The adult feeding behavior of Aplysia develops in a stereotyped fashion (Kriegstein et al., 1974). Although the consummatory response (functional rasping) emerges only at the end of metamorphosis, the appetitive head-waving response of the adult feeding pattern is already present at metamorphosis. If dislodged from the seaweed, metamorphic animals with distinct anterior tentacles can be seen to anchor their metapodium on a substrate and wave their heads and bodies from side to side in an orienting manner (Kriegstein et al., 1974). However, this overall response could also serve as a general searching behavior that can occur independent of feeding, aiding, for example, in finding the appropriate metamorphic substrate. As mentioned earlier. in response to external disturbances, the veliger exhibits a defensive response characterized by the withdrawal of the cephalopedal mass into the shell and thc closing of the shell aperture with the operculum. This withdrawal reflex is still present during thc early phase of metamorphosis (Kandel, 1979). However, subsequent growth of the cephalopedal mass prevents successful withdrawal inside the shell and the animal often responds to tactile stimulation of the head with generalized body contraction. It may also be that the larval retractor muscles are no longer functional for withdrawal at this stage. With further growth, the withdrawal responses become progressively restricted to the stimulated body parts (Kandel, 1979).

Hadfield, 1977) and in other opisthobranchs (Bonar and Hadfield, 1974; Bonar, 1978a; Bickell and Kempf, 1983), at least some of these cells are ingested, whereas in other species the cells are lost. lysed, or phagocytosed (Thompson, 1958: Tardy, 1970; Thiriot-QuiCvreux, 1970; Bridges, 1975; Kempf and Willows, 1977; Bonar. 1978a; Morse et al., 1980). In many prosobranchs, the velar lobes are shed by a violent contraction of the retractor muscles and are subsequently sucked into the mouth as the first meal (Fretter, 1972); all of this happening within a matter of seconds! The velar tissue of bivalves also disappears at metamorphosis (D’Asaro, 1967; Bayne, 1971 ; Hickman and Gruffydd, 197 1 ), probably by phagocytosis (Bayne, I97 1; Hickman and Gruffydd, 1971 ). In aplysiids, gradual resorption of the remaining velar tissue ensues until two stubby lobes that form the primordia of the anterior (cephalic) tentacles develop (Fig. 2 ) (Bridges, 1975; Kriegstein, 1977a; Switzer-Dunlap and Hadfield. 1977). In other opisthobranchs, the remnants of the velar lobes are incorporated into the head epidermis or fuse and grow anteriorly to contribute to oral veil or oral hood formation (Bonar, 1978a; Bickell and Kempf, 1983). In aplysiids and other opisthobranchs. the later phases of metamorphosis are characterized by onset of adult heart function, atrophy of the larval heart, resumed crawling, growth and reorganization of the foot, mantle and velar lobe rudiments, as well as development of the adult digestive and excretory systems, and onset of radular or buccal activity (Kriegstein, 1977a; Switzer-Dunlap and Hadfield, 1977; Paige, 1988; Bonar, 1978a). Metamorphosis is terminated with the onset of functional feeding. The animal has thus completed its shift from a pelagic to a benthic life-style. However, further body growth and morphogenesis occur during the long juvenile period, exemplified by the formation of siphon, gill, parapodia, and rhinophores ( Kriegstein, 1977a; Switzer-Dunlap and Hadfield, 1977; Bickell and Kempf, 1983; Rankin et al., 1987: Paige, 1988).

Morphogenetic Response The first stage of metamorphosis involves the loss of velum. An early indication of this upcoming event is a gradual asynchrony of the normally metachronal ciliary beat (Bonar and Hadfield, 1974; Bridges, 1975), perhaps as a result of electrical uncoupling between the senescent velar cells (Arkett, 1988). These ciliated cells are subsequently shed. In aplysiids ( Switzer-Dunlap and

Muscular and Sensory Alterations during Metamorphosis The larval retractor muscles undergo dramatic reorganization during metamorphosis. In nudibranchs ( Opisthobranchia) , these muscles degenerate and are autolysed soon after shell loss (Thompson, 1958, 1962; Tardy, 1970; Bonar and Hadfield, 1974; Bonar, 1976, 1978a; Bickell and

Gutropod Meiamorphosis

Kempf, 1983). The contraction of these muscles, prior to their destruction, seems to be involved in shell extrusion and body flattening in these same species (Tardy, 1970; Bonar and Hadfield, 1974; Bonar, 1976; Bickell and Kempf, 1983). In the shelled opisthobranchs, these muscles may persist but are modified to subserve new functions (Bonar, 1978a). In the cephalaspid Retzisa ohtusa, however, they degenerate and the adult columellar muscle develops independently (Smith, 1967). The fate of the larval retractor muscles in aplysiids has not been determined. Juvenile Phjdiaplysia have 11 muscles originating from the shell that insert into the veliconch body; but how or if these muscles originate from the two larval retractor muscles is unknown (Bridges, 1975). In prosobranch gastropods, the fate of the larval retractor muscles is complex: some of them degenerate, whereas other parts are preserved but modified to form local muscles associated with the buccal mass or with the cephalic mass: still others form the large adult columellar muscle (Fretter, 1969, 1972). In bivalves, the velar retractor muscles atrophy or degenerate altogether ( D’Asaro, 1967; Bayne, 1971; Hickman and Gruffydd, 1971; Moor, 1983). The powerful bivalve adductor muscles arise independently, generally prior to metamorphosis. The circular, longitudinal, and oblique cells of the diffuse subepidermal muscle system usually persist through juvenile life, except for those associated with organs lost at metamorphosis (e.g., the velum) (Thompson, 1958, 1962; Bonar and Hadfield, 1974; Bonar, 1976). Counteracting this widespread muscular atrophy is a period of renewed myogenesis associated with the development of organs, especially towards the end of metamorphosis. Thus muscles associated with the buccal mass, foot, adult heart, tentacles, and body wall. as well as the large columellar or adductor muscles proliferate as these diffcrent tissues grow (Bonar, 1976, I978a; Moor, 1983; Bickell and Kempf, 1983; Bayne, 1971). Little change in sensory structure occurs during metamorphosis of opisthobranchs. Both statocysts and eyes are present prior to and persist through metamorphosis (Kriegstein, 1977a: Switzer-Dunlap, 1978; Bonar, 1978a; Paige, 1988). The anlenor (oral) tentacles of aplysiids take shape towards the end of metamorphosis (Kriegstein. 1977a: Switzer-Dunlap and Hadfield, 1977; see above). The fate of the apical (cephalic) sensory organ is not clear. Tardy (1970) mentions that this structure persists until metamorphosis. This structurc is

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still present and in contact with both the cerebral ganglia and the overlying epidermis in the 2-day postlarvae of I‘hestillu (Bonar, 1978a,b). Bonar ( 1978a) suggests that the cells of this structure may be incorporated in the developing cephalic tentacles. However, the homologous structure in bivalves disintegrates after metamorphosis; it is the surrounding cells of the apical plate that are integrated in the labial palps (tentacular structures) (D’Asaro, 1967; Bayne, 1971; Hickman and Gruffydd, 197 1 ).

Neuronal Reorganization during Metamorphosis Ganglionic Reorganization. Neuronal reorganization during metamorphosis has been observed at the gross level in many species. In Aplysia californica, it consists only of a relative rearward migration of the cerebral, pedal, and pleural ganglia from a position anterior to the buccal mass before metamorphosis, to a position surrounding the buccal mass after metamorphosis. This migration is accompanied by a lengthening of commissures and connectives (Fig. 3 ) (Kriegstein. 1977a,b). In the nudibranch Mefihe leonina, metamorphosis is characterized by a similar lengthening of the eommissures and connectives of the central nervous system, and a centrifugal migration of the rhinophoral ganglia from their ipsilateral cerebral ganglia (Bickell and Kempf. 1983). In the nudibranch Aeolidiella ulderi, which undergoes metamorphosis at the end of its capsular development, the central nervous system undergoes two types of transformations (Fig. 3 ) : ( 1 ) cerebralization, which consists of anterior migration and fusion of the ganglia of the visceral loop with the cerebropleural ganglia; and (3) telencephalization, which is an invagination from the intravelar area that detaches and fuses with each cerebropleural ganglion (Tardy, 1970, 1974). In bivalves, the larval nervous system appears to be as extensive, if not more so, than in postmetamorphic animals (D’Asaro, 1967; Bayne, 1971; Hickman and Gruffydd, 197 1 ) . This is especially true of the oyster Ostreu eduli;\ whose visceral ganglion is the only one to grow during the transition from the larval to the postmetamorphic stages, whereas the cerebral and pedal ganglia, as well as the eyes and foot. atrophy considerably during the same period ( Hickman and Gruffydd, 197 1 ) . A significant morphogenetic event during metamorphosis in opisthobranchs is detorJion of the visceral mass (Tardy. 1970; Kriegstein, 1977a;

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Murois und Curew LARVAL OPISTHOBRANCH

VISCERAL LOOP

I

METAMORPHOSIS & JUVENILE DEVELOPMENT

APLYSIA

AEOLlDlELLA

Figure 3 Diagrammatic representation of a dorsal view of the larval and juvenile CNS of two opisthobranch species. From a similar CNS structure at larval stage, differential forces initiated at metamorphosis lead to the pronounced anatomical divergence characteristic of juvenile opisthobranchs. There are slight variations among opisthobranch species in terms of the number and identity of ganglia associated with the visceral loop (shaded area). In A p l y ~ i a ,starting at metamorphosis there is an elongation of the commissures and connectives of the CNS, as well as a slight detorsion of the visceral loop. In Aeolidiella, two movements of neuronal masses are initiated at metamorphosis: ( 1 ) Telencephalization leads to thc fusion of an invaginated mass from the intravelar area into the anterior portion of the cerebro-pleural ganglion; ( 2 ) cerebralization consists chiefly of the anterior migration and fusion of the ganglia of the visceral loop with the cerebro-pleural ganglion. The buccal ganglia (BG), absent for the most part of larval life, form just prior to metamorphosis. CG = cerebral ganglion; PG = pedal ganglion.

Bonar, 1978a). This process reverses, to various extents for diffcrcnt specics, the torsion that the visceral mass undergoes during embryogenesis. In hatchling Aplysia, the visceral mass is twisted about 120” (Saunders and Poole, 19 10). The ganglia of the visceral loop escapc torsion, although not completely, because they develop during the larval period anterior to the affected region

(Tardy, 1970, 1974; Kriegstein, 1 9 7 7 4 . Kriegstein ( 1977a)argues that the ganglia of the visceral loop are not affected by detorsion as “they move from the head region (not involved in detorsion) into the more posterior visceral region (which is now detorted) only after detorsion is complete.” However, Marois and Carew (in preparation) observed that the visceral loop, as detected by serotonin immunohistochemistry, is considerably twisted (more than I 00O) at the end of the larval phase. We conclude that the visceral ganglionic loop migrates into the still torted visceral region and that it is subsequently untwisted (but not completely) by detorsion during metamorphosis.

Cellular Reorganization. From a cellular standpoint, the elaboration of thc nervous system during metamorphosis has been almost exclusively studied in opisthobranchs. The neuropil of all the central ganglia enlarges considerably during metamorphosis (Kriegstein, 1977b; Schacher et al., I 979b: Bickell and Kcmpf, 1983), The volume of individual nerve cells also increases during this pcnod (Kriegstein, 1977b; Schacher et al., 1979b; Bickell and Kempf, 1983). As a result of this massive growth in cell body size, a few identified neurons can be reliably distinguished in the CNS for the first time at metamorphosis (Kriegstein, 1977b; Bickell and Kempf. 1983). The enlargement of the central nervous system is not only due to the growth of individual cells but also to the addition of new elements (Tardy, 1970, 1974; McAllister et al., 1983; Jacob, 1984). The origin of thcse neurons appears to be similar to the pattern observed in larval stages; presumptive neurons proliferate from spccific, laminated ectodermal zones of the body wall and migrate. postmitotically, into adjacent ganglia (Smith, 1967; Tardy, 1970, 1974; Guyomarc’h-Cousin, 1974; McAllister et al., 1983; Jacob, 1984). Only glial proliferation is presumed to occur within the CNS (Jacob, 1984). This neurogenic pattern does not end with metamorphosis, but continues throughout the juvenile growth of the animal (McAllister et al., 1983; Cash and Carew, 1989; Hickmott and Carew, 1988: in preparation). In fact, numerous peripheral ganglia originate after metamorphosis (Tardy, 1970, 1974; Kriegstein, 1977a,b; Bickell and Kempf, 1983). Metamorphosis is a dual process involving both elaboration of adult structures and elimination or reorganization of larval tissues. Although the central nervous system shows obvious signs of growth during metamorphosis. little evidence has yet been

Gastropod Metamorphosis

accumulated for regressive events in this tissuc. The sensory and especially the muscular alterations occurring during metamorphosis alone strongly suggest that the nervous system must also undergo reorganization. For instance, for both opisthobranchs (Carter, 1926; Kempf et al.. 1987. Marois and Carew, 1989, in preparation) and prosobranchs (Mackie et al., 1976; Arkett et al., 1987; Arkett, 1988), nerve fibers presumed to originate from the cerebral ganglion appear to innervate the ciliated cells and muscle fibers of the velum. Because the velum is lost at metamorphosis these nerve cells must reorganize their projections or simply be eliminated. Although, to our knowledge, innervation of other transitory larval structures has not been examined, it seems likely that neuronal reorganization must occur elsewhere as well. The high coordination and speed of contractility of the larval retractor (and cephalopedal) musculature, together with the fact that neuromuscular antagonists such as succinyl chloride and procaine hydrochloride produce relaxation or paralysis in opisthobranchs (Bonar, 1976; Kriegstein, 1977a), suggest that these muscles arc also under neural control. The effector neurons subserving these muscles should then also be affected by metamorphosis. To dctermine the impact of metamorphosis at the level of single identifiable cells in the nervous system of Aplysia calijornica, we have recently used immunocytochemical techniques to selectively label serotonergic neurons in the embryonic, larval, and metamorphic stages of development (Marois and Carew, 1989, in preparation). The serotonergic pattern in the cerebral ganglia of competent larvae Of AjdyYia consists of four bilaterally symmetric pairs of cells and an unpaired median cell (Fig. 4 ) . As early as the embryonic stage, this median cell, as well as one of the bilateral pair of neurons, sends anterior radiating projections into the velum. Concomitant with the early phase of metamorphosis (loss of the velum), the velar projections of the bilateral pair are lost, whereas the unpaired median cell disappears altogether. We do not know yet whether this cell simply alters its transmitter phenotype (i.e., is no longer serotonergic and thus cannot be detected) or undergoes cell death. However, at present we favor the latter hypothesis since several small serotonergic globular elements, reminiscent of a disintegrative or phagocytic process. have on a few occasions been observed in lieu of the soma of the unpaired median cell. During the late phase of metamorphosis, which is marked by the onset of

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Figure 4 The effect of metamorphosis on the serotonergic cells of .4idy~iacul(/ornica. Diagrammatic representation of a dorsal view ofthe anterior half of a metamorphosing veliger. The shaded circles indicate the approximate location of cerebral ganglia. although their exact boundaries are unknown. (A) Competent larva. There is a bilaterally symmetrical cluster of four serotonergic cells in the cerebral ganglia. Two of these cells send anterior varicose projections into the velum and medial projections in a ncuritic plexus. An unpaired median cell sends similar projections to the velum. Other projcctions originating from the cluster of serotonergic cells extend elsewhere in the CNS and in the periphery (not shown here). ( B ) Early phase of metamorphosis. Concomitant with the loss of the velum, the unpaired median cell and all velar projections disappear while the projections into the neuritic plexus persist. ( C ) Late phase of metamorphosis. A fifth cell is added to each cluster. Another bilaterally symmetric cell appears medial and anterior to the cluster. This cell can later be recognized as the metacerebral giant (MCG or C1) cell, although its cercbro-buccal projection is not visible at this stage (Marois and Carew, in preparation.)

I044

Maruis and Chrrw

radular function, a well-studied bilateral pair of serotonergic neurons involved in the feeding motor program (the metacerebral giant or C 1 cells) are detected for the first time (Fig. 4). Thus at lcast two principles of neuronal reorganization at metamorphosis are illustrated by the changes in the serotonergic pattern: ( 1 ) remodelling of axonal projections; and ( 2 ) cell addition. To address whether cell death is a third principle of neuronal reorganization in gastropods, we are presently trying to determine the fate of the unpaired median cell. The occurrence of cell death is of considerable theoretical importance given that, in contrast to most animal groups in which cell death plays a major role in development (Truman, 1984; Clarke, 1985), there are presently no reports of nerve cell death in molluscs, either during metamorphosis nor at any other stage of development (Jacob, 1984: Cash and Carew, 1989; Croll and Chiasson, 1989).

Possible Mechanisms Underlying Neurometamorphosis Given the dramatic neuronal and nonneuronal alterations that we have described during metamorphosis, a key question arises concerning the factor( s) that mediate this striking reorganization. Are they principally hormonal, as in amphibians and holometabolous insects (Fox, 1984; Balls and Bownes, 1985; Truman et al., 1985; Weeks and Levine, 1990)? Are they principally neurally mediated, as may be the case with the sea urchin larvae Dendraster excentricus (Burke, 1983)? Or is some combination ofthese or other mechanisms involved? Although the underlying mechanisms of the metamorphic process in gastropods are largely unknown. a role for neurosecretory products in affecting nerve cell growth during metamorphosis in Aply.sia has been postulated by Schacher and colleagues ( 1979b). They identified in the central ganglia of the Aplysia nervous system, a class of nonneuronal support cells which contains secretory granules. The contents of these granules are normally released at metamorphosis, when a large increase in cell body growth and synaptic density occurs. The selective premature release of this granular material during midlarval phase can be brought about by high-K+/low-Ca2+artificial seawater, and results in an early increase in nerve cell body size, spine formation, and synaptic density. Interestingly, these nonneuronal support cells subsequently take on the morphology of adult glial

cells. The secretory material in Apl.vsia was tentatively identified as proteinaceous ( Schacher et al., 1979b). Another secretory product putatively involved in gastropod metamorphosis is present in the apical sensory organ of the prosobranch Huliolis rz&scens (Morse et al., 1980). This material, possibly a glycopeptide, can be released from the secretory cells of this sensory organ by the metamorphic inducer gamma-amino butyric acid (GABA), and is thought to be necessary for subsequent morphological metamorphosis (Morse et al., 1980). Another interesting parallel between Aplysia and Haliotis is that treatment with high K + seawater induced some metamorphic changes in both species, suggesting the involvement of excitable cells in this process. In Huliotis, high-K+/ normalCa2+ seawater (which triggers vesicular release produced by depolarization of excitable cells) induces complete mctarnorphosis, but high-K+/ low-Ca2+ (which reduces or eliminates vesicular release produced by depolarization) does not (Baloun and Morse, 1984). In their study ofAplysia, Schacher et al. ( I979b) used high-K+/low-Ca2+ seawater instead of high-K+/ normal-Ca" to induce selective secretory release from the nonneuronal support cells in the central nervous system (CNS) because the latter solution leads to additional release of material from other secretory gland cells, such as those in the propodium or body wall. Their study did not examine whether high-K+/ normal-Ca2+seawater induces complete metamorphosis in competent Aplysia larvae. Nevertheless, different ionic requirements for release from different secretory systems may explain why the high-K'/low-Ca'+ seawater fails to induce metamorphosis in Haliotis, whereas high K / normal-Ca2+ is effective. It may be that the lowCa2+medium does not promote release from other critical secretory systems involved in metamorphosis, such as the apical sensory organ. If Aplysia larvae behave as Haliotis larvae do, and can be induced to metamorphose in normal (but not low) Ca2+ levels in high-K+ seawater, then they may provide a means of examining the interaction of the nervous system with its peripheral target tissues during the metamorphic process. For example, the neuronal alterations associated with metamorphosis, as observed with serotonin immunohistochemistry (Marois and Carew, in preparation) may not necessarily result from a direct effect of metamorphic factors on the CNS; the nervous system may primarily be responding to the ongoing morphogenetic changes occumng in

Gustropod Metamorphosis

the peripheral tissues which, in turn, could induce changes in the CNS. In principle, one could examine the role of the granular material released from the nonneuronal support cells in the absence of peripheral target changes by comparing the effect of high-K+/low-Ca*+seawater (which should selectively affect the CNS (Schacher et al., 1979b)) with the effect of high-K+/normal-Ca2+ seawater (which should affect both the CNS and peripheral tissues) on the pattern of serotonergic cells in Aplysia larvae.

Competence and Induction Competence for metamorphosis is indicated in Aplysia culifornica by the appearance of four to six red spots on the right side of the outer perivisceral membrane and a red band overlying the anterior mantle margin (Kriegstein, 1977a). However, in most other opisthobranchs, no such obvious external marker exists and the acquisition of competence, reached sometime after propodial development, must be determined empirically (Bonar and Hadfield, 1974; Kempf and Willows, 1977; Switzer-Dunlap and Hadfield, 1977; Hadfield, 1978; Switzer-Dunlap, 1978; Bickell and Chia, 1979; Bickell and Kempf, 1983; Paige, 1988). The development of the benthic loconlotory organ, the foot, seems to be a useful marker of metamorphic competence in prosobranchs and bivalves as well ( D’Asaro, 1967; Fretter, 1967, 1969; Hickman and Gruffydd, 1971; Boyle and Turner, 1976). Once the free-swimming veliger of most opisthobranchs and many other molluscs is competent to metamorphose, it will undergo no further development unless presented with a triggering substrate for metamorphosis (Hadfield, 1978; Morse, 1985; for exceptions see D’Asaro, 1967; Franz, 1971 ; see also below). Competent aplysiid larvae deprived of the triggering substance for weeks or months seem to be in a state of arrested growth; they simply maintain a normal larval lifestyle, feeding and swimming sporadically until an appropriate settling substrate or substance becomes available (Kriegstein et al., 1974; Switzer-Dunlap, 1978; Paige, 1988).

Nature and Action of Inducing Substances Inducing substances appear to be extremely varied. They can be associated with the food source (animal or plant) of the adult, with bacterial and other microorganismic populations present in the

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adult environment, with the presence of other conspecifics (or tissues of conspecifics), and with specific types of sand, mud, or wood (Thompson, 1958, 1962; Tardy, 1970; Kriegstein et al., 1974; Kempf and Willows, 1977; Switzer-Dunlap and Hadfield, 1977; Chia and KOSS,1988; Hubbard, 1988; Fitt et al., 1989; for reviews see Crisp, 1974; Hadfield, 1978). The natural inducing substances may either be dissolved in seawater or substratebound (Hadfield, 1978; Coon et al., 1985; Morse, 1985; Fitt et al.. 1989). Solubilized extracts derived from the settling substrate can also induce metamorphosis ( Hadfield, 1978: Morse, Hooker, Duncan, and Jensen, 1979; Fitt et al., 1989). For aplysiids, each species examined appeared to settle and metamorphose preferentially on a few species of macro- or bluegreen algae which, not surprisingly, make up a large portion of the adult diet in the natural habitat. (Kriegstein et al., 1974; Switzer-Dunlap and Hadfield, 1977; Paige, 1988; but see Pawlik, 1988) That the inducing factors are typically associated with the habitat of the adult animal presumably insures that the veliger can metamorphose in an environment suitable for its juvenile growth and survival. Correspondingly, species that feed on multiple prey items or on prey with ubiquitous distributions in time and space, such as the nudibranchs Melibe leonina and Tritonia diumedea, appear not to need any specific inducers for metamorphosis (Kempf and Willows, 1977; Bickell and Kempf, 1983). Finally, as one would expect, the requirement for an inducing substrate is absent in species like PhyllapI.ysia that undergo metamorphosis inside the egg capsule (Bridges. 1975) . Inducing substances probably vary in their mode of action. For instance, seawater containing GABA, or a natural GABA-like substance associated with the natural recruiting algae, is sufficient to trigger both normal settlement and metamorphosis of larvae of the prosobranch Huliolis (Morse et al., 1979; Morse et al., 1980; Morse and Morse. 1984a,b; see below). It is not known in this case if the GABA compound triggers independently the settling and metamorphic processes or if it activates only settlement, which in turn would elicit metamorphosis. On the other hand, it has been suggested that for the nudibranch Onchidoris and the oyster Crasmstrea, there may be a diffusible chemical emanating from the substrate involved in larval settlement, whereas another substrate-bound chemical cue or mechanochemical cue is responsible for inducing metamorphosis (Coon et al., 1985; Chia and Koss, 1988; Fitt et al.,

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JVfaroisand Careit.

1989; see below). In any case, that the natural inducers are simply acting as triggers of the metamorphic process, rather than mediators of the entire metamorphic event, is suggested by the fact that brief exposure of the larvae to the substance is often sufficient to induce settlement and the subsequent morphogenetic transformations ( Hadfield, 1978; Coon et al., 1985). The Nervous System in the Induction Process

Detection of the inducing stimulus in marine invertebrate larvae can involve visual, tactile, and most frequently, chemical stimuli (for reviews see Crisp, 1974; Hadfield, 1978). The sensory basis of this induction process seems to suggest the involvement of the nervous system in settlement and metamorphosis (Hadfield, 1978; Burke, 1983). Lending support to this hypothesis are the findings that ( 1 ) some of the molluscan morphogenetic chemical cues appear to be neurotransmitter-like substances (Morse et al., 1979: Weiner and Colwell, 1982: Cooper, 1982: Coon et al., 1985; Morse, 1985), and ( 2 ) some molluscan species metamorphose when presented with specific neurotransmitters or related chemical compounds (Bonar. 1976; Hadfield, 1978: Morse et al., 1979). For example, at high concentrations and long exposure, many forms of choline (but not acetylcholine) induce metamorphosis in various opisthobranchs (Bonar, 1976; Hadfield, 1978; Harrigan and Alkon, 1978; Hirata and Hadfield, 1986). Also, GABA acts potently on the prosobranch Haliotis Rufescens (Morse et al., 1979; Morse et al., 1980) and less so on the opisthobrancli Phestilla sihogue (Hadfield, 1984). Moreover, in the pacific oyster C’r-ussostreagigas, L-Dopa (but not dopainine) induces both settlement and metamorphosis, whereas norepinephrine and epinephrine as well as a number of adrenergic agonists only induce metamorphosis (Coon et al., 1985; Coon and Bonar. 1987). In addition, various catecholamincs lead to partial metamorphosis (loss of velum) in Phestilfa (Hadfield, 1984). That these effects are compound-specific is suggested by the fact that compounds structually related to the aclive ones have no effect. In most cases, it appears unlikely that these transmitter-like chemicals represent the natural inducer. For example, choline is not thought to be the substance in the coral extract that induces metamorphosis in the opisthobranch Phestillu; the dosages, and action mode appear to differ for the

two compounds (Hirata and Hadfield, 1986). Moreover, the metamorphogenetic response of Phestillu to chemical inducers has different kinetics than those associated with the coral-produced inducing substance, and some of these chemicals induce only localized transformations (e.g., loss of velum) (Hadfield, 1984). Likewise, Fitt et al. ( 1989) recently reported that the as yet unidentified bacterial substance that induces settlement behavior of veliger larvae of the oyster C. gigas is probably not L-Dopa or a melanin-like polymer of L-Dopa. It may be that these chemical compounds are instead involved in the central neural response to the natural inducer (Hirata and Hadfield, 1986). In fact, dopamine (and norepinephrine) have been detected in appreciable quantity in the larvae of the pacific oyster (Coon and Bonar, 1986). In contrast to the above examples, in a few cases the natural inducing substance does closely resemble a neurotransmitter. For instance, it seems likely that the natural inducer of larval settlement and metamorphosis of many species of the abalone ( Prosobranch) Haliotis is a GABA-like peptide present on the surface of the crustose red algae (Morse et al., 1979, 1980; Morse and Morse, 1984a,b; for review see Morse, 1985). The transducing response of the veliger to the inducing substance is not yet clear. but Morse and colleagues have accumulated evidence for the following hypothesis: The binding of the inducing ligand to specific receptors on the surface of the larva triggers changes in CAMP concentration and C1- efflux from the chemosensory cells, leading to their depolarization (Morse et al., 1980; Baloun and Morse, 1984; Morse, 1985; see also Baxter and Morse. 1987). This depolarization is then conveyed to the larval nervous system, which ultimately gives rise to the metamorphic response in the target tissues. In fact, exposure of competent Haliotis larvae to high-K+ seawater, a treatment that leads to the depolarization of excitable cells, elicits metamorphosis (Baloun and Morse, 1984). The nature and location of the presumed chemosensory organ involved in transduction of the metamorphic signal is unclear. A strong candidate is the apical sensory organ present on many opisthobranchs (such as Aplysia) and other gastropods (Tardy, 1970; Bonar, 1978a.b; Mone ct al., 1980), as well as bivalves (D’Asaro, 1967; Bayne, 197 1 ; Hickman and Gruffydd, 197 1 ). In the nudibranch Phestillu sihogae where it has been best studied, this organ contains superficial ciliated cells with chcmosensory ultrastructure (Bonar,

Gastropod !Mctamorphosis

1978a,b). The axons of these cells project into the underlying cerebral commissure and cerebral ganglia. Interestingly, the position of this organ is well suited for sampling of the water currents created by the velar ciliary action, and it stands very near the substrate during the inspection of settling sites by a competent larva (Bonar and Hadfield, 1974). The chemosensory structure of this organ and its close association with the cerebral ganglia have also been reported in bivalve veligers (Bayne. 1971: Hickman and Gruffydd, 1971 ) . It would be interesting to examine whether the equivalent organ in Huliotis bears the chemosensory reccptors for the GABA-like inducer. That thc nervous system is directly involved in mediating transduction of the morphogenetic signal of a marine larva has been demonstrated in the sand dollar ( echinoid) Dendraster Excentricus (Burke, 1983). Electrical stimulation of a sensory apical region of neuropil near the mouth of the animal leads to metamorphosis. Burke (1983) proposed that the apical neuropil and the associated oral ganglion of the echinoderm transduce the morphogenetic signal via chemoreceptors into an excitatory neural response that stimulates tissues to initiate the sequence of developmental events of metamorphosis. In addition, the larvae of one annelid and three molluscan species can be induced to metamorphose by exposure to high-K' seawater, presumably via the depolarization of excitable cells involved in this process (Yool et al., 1986). It may thus be that the involvement ofthe nervous system in settlement and metamorphosis is ubiquitous to free-swimming marine larvae. Maturation of Competence Regardless of the nature of the specific factors involved in the metamorphic response, it seems evident that metamorphic competence is not acquired by the maturation of receptors for the inducing substance, as premature exposure of both Huliotis and Phestilla larvae to the inducer results in desensitization of the competent larvae to the compound (Hadfield, 1984; Hirata and Hadfield, 1986; Trapido-Rosenthal and Morse, 1986a) and in addition, for Ilafiotis at least, in receptor downrcgulation ( Trapido-Rosenthal and Morse, 1986b). Based on the study by Schacher and colleagues ( 1979b) in Aplysia, we also know that the target tissue (in this case, neurons and their support cells) can, in precompetent larvae, undergo alterations normally associated with metamorphosis. Therefore. competence seems to be acquired

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by the maturation of one or more steps along the pathway between sensory reception ( i.e., chemosensory receptors) and the receptive target tissue (i.e., neuronal and nonneuronal support cells). Some insights into the nature of the pathway might be gained by comparing the onsets of metamorphic competence to the natural inducer on the one hand and to the high-Kf/ normal-Ca2+ medium on the other.

CONCLUDING REMARKS

Several lines of evidence described in this review support an activc role for the nervous system in mediating the metamorphic response of freeswimming gastropod larvae: ( 1 ) Both neurotransmitter-like natural inducers and synthetic compounds can induce metamorphosis; ( 2 ) Natural inducers seem to act via chemosensory receptors; ( 3 ) The apical sensory organ, a transitory larval structure, is believed to transduce the induction signal to the central nervous system, probably within the cerebral ganglia; ( 4 ) Conditions that lead to depolarization of excitable cells in the veliger induce metamorphosis; and ( 5 ) Appropriate neurally-mediated behavior is necessary for both settlement and metamorphosis to proceed. From the CNS, information is distributed in at least two possible pathways. The first may be purely neuronal; it is specific, fast, and responsible for the behavioral response of the animal to the inducing signal, such as settlement and the coordinated muscular activation necessary for detachment of the shell and/or velum. It is also reversible as the animal will not proceed through metamorphosis if disturbed. The second pathway may be neuronal and/or hormonal. It is probably widely distributed to the target tissues and leads to all the slower morphological (and behavioral ) alterations associated with metamorphosis; it is also irreversible. An interesting avenue of future research would be to examine if the tissue involvcd in transduction of the metamorphic signal (for example, the apical sensory organ) has input to the animal's neuroendocrine centers, analogous to those characterized in the cerebral ganglion of the pulmonate Lymnaen (Joosse. 1986). The involvement of the nervous system in metamorphosis of free-swimming molluscan larvae appears to be distinct from that of holometabolous insect and amphibian larvae. Metamorphosis in the latter animals seems to be triggered by an internal state (such as body weight for insects, and

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development of specific brain nuclei for amphibians), and the environment plays a role in so far as it is permissive to the event (Granger and Bollenbacher, 1981; Fox, 1984; Rosenkilde. 1985; Truman et al., 1985). By contrast, the environment is instructive to the molluscan larvae since they must exert the appropriate sensory and motor responses to a specific external stlmulus (inducing substance) for metamorphosis to proceed. On the other hand, once the metamorphic process is set in motion, the response of the molluscan nervous system to the induction process appears similar to that described for holometabolous insects and amphibians. The processes of nerve cell growth, cell addition, and axonal reorganization observed during Aplysza metamorphosis also occur in these other animals (Alley and Barnes, 1983; Barnes and Alley, 1983; Fox, 1984; Truman et al., 1985; Weeks and Levine, 1990). However, unlike insects and amphibians, cell death is a scarce phenomenon (if present at all) in molluscan development, even during metamorphosis. If cell death does occur in molluscs, it may be more likely to be used in bivalve larvae that undergo a drastic ganglionic reorganization during metamorphosis (Hickman and Gruffydd, 197 1 ). When surveying the developmental pattern of molluscan species with free-swimming molluscan larval phases, it is striking that there are three functionally distinct nervous systems in the life history of the animal. Each of these nervous systems is adapted for a unique lifestyle that requires a distinct set of sensory and motor functions. First, there is the larval organization of the nervous system, responsible for planktonic feeding. dispersal and a pelagic life-style. Second, there is a specialized metamorphic nervous system, responsible for substrate searching, settlement and metamorphosis. Finally, there is the juvenile nervous system responsible for an herbivorous or carnivorous nutritional mode, benthic life-style and, ultimately, reproduction. Although each of these nervous systems has unique features and serves unique functions, their life histories overlap, and they all share many common neural traits. We have just begun to understand how these neural systems differ from each other. Moreover, the question of how the transitions between each of these systems are brought about is still unanswered. Thus in the field of molluscan developmental neurobiology, the study of metamorphosis is still in its larval stage. We are grateful to our colleagues Emilie Marcus, Mark Stopfer, and William Wright for their helpful

comments and suggestions. We are also indebted to David Cook for his skillful artwork on the figures. The preparation of this review and some of the work presented in it was supported by an NSERC (Canada) fellowship to R.M. and by NSF grant HNS 83 l 1300 and ONR contract NO00 14-87-K-0381 to T.J.C.

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