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Development of embryonic and larval cells containing serotonin, catecholamines, and FMRFamide-related peptides in the gastropod mollusc Phestilla sibogae.

Introduction

Our understanding of early neural development in gastropod molluscs has increased greatly over the last decade. Much of the recent work on this topic has relied upon histochemical techniques to visualize cells as they first acquire their transmitter contents and elaborate their innervation of peripheral tissues (for reviews, see Croll, 2000; Croll and Dickinson, 2004; but also see references listed below). Fortunately, these studies have also been able to build upon a solid foundation of previous and concurrent electron microscopic studies (Chia and Koss, 1984; Page, 1992a, b; Marois and Carew, 1997a; Page and Parries, 2000). Through such work we now have descriptions of larval nervous systems in heterobranch, caenogastropod, and vetigastropod molluscs. Together, these studies consistently indicate that the nervous system first appears in the early trochophore stage and continues to add components through subsequent veliger stages. The work also indicates that a large portion of the larval nervous system lies outside the ganglia that eventually develop into the adult central nervous system (i.e., the cerebral, pedal, pleural, parietal, and visceral ganglia).

The nudibranch gastropod Phestilla sibogae Bergh has played a prominent role in the study of larval development in molluscs. Members of this aeolid species are abundant in tropical localities and easily reared in the laboratory, where they normally require 5 days of embryonic development and another 3 to 4 days of larval development before becoming competent to metamorphose into juvenile sea slugs (Miller, 1993). Given these advantages, it is not surprising that P. sibogae was the subject of one of the earliest ultrastuctural descriptions of the apical sensory organ (Bonar, 1978), which is a major component of the larval nervous system in gastropods (Croll and Dickinson, 2004). In fact, P. sibogae was also one of the earliest species to be examined using immunocytochemical techniques for the identification of larval neurons (Kempf et al., 1992).

However, despite the increasing numbers of studies focused upon neural development in gastropods, many questions remain. For instance, our knowledge of the larval nervous system in P. sibogae, as in most other species, is based upon only a very limited range of larval ages. Kempf et al. (1992, 1997) and Pires et al. (2000a), studying the distributions of a variety of neurotransmittters, all examined only late larval stages. Little is currently known of when specific nerve cells first appear during the larval development of this species or any other nudibranch gastropod (but see Buznikov et al., 2003). Furthermore, while P. sibogae has been the focus of numerous studies (Hadfield, 1978, 1984; Hadfield and Scheuer, 1985; Miller and Hadfield, 1986; Hadfield and Pennington, 1990; Pires and Hadfield, 1991; Hadfield et al., 2000; Leise and Hadfield, 2000; Pires et al., 2000a) into the role of the nervous system in triggering metamorphosis, little is known about changes within the nervous system that correlate with the onset of metamorphic competence.

The present study examines the distribution of neurons exhibiting serotonin (5-HT)- and FMRFamide-like immunoreactivities in embryonic and larval stages of P. sibogae. The distribution of catecholaminergic neurons was also examined through the use of an antibody raised against tyrosine hydroxylase (TH), the enzyme that catalyzes the conversion of tyrosine to DOPA, the initial step in catecholaminergic synthesis (see Cooper et al., 1996; Pani and Croll, 1998; Pires et al., 2000a). In this study I provide details of the locations and morphologies of the various immunoreactive neurons. I also provide a detailed account of the ontogeny of various neurons from the time they first exhibit immunoreactivity until the time that the larvae reach metamorphic competence. Previous evidence indicates that such cells may be present long before they contain detectable levels of transmitters, but transmitter content may be a better indicator of functionality and thus provide better insight into the roles of the nervous system in controlling larval behavior and physiology. Finally, with the recent publication of descriptions of serotonergic, catecholaminergic, and peptidergic neurons and fibers in both the central and peripheral nervous systems of P. sibogae (Croll et al., 2001, 2003), the present study also sets the stage for future detailed studies into changes in the nervous system as this gastropod undergoes metamorphosis.

Materials and Methods

Animals

Individuals of Phestilla sibogae were collected from the wild and maintained in Honolulu in outdoor flow-through seawater tables under ambient light and temperature. Animals were fed live pieces of the coral Porites compressa and eggs were collected daily. The embryos and hatched larvae were maintained at about 26[degrees]C with constant agitation and aeration, as described previously (Miller and Hadfield, 1986).

Immunocytochemistry

Wholemount immunohistochemistry employed procedures modified from Croll and Chiasson (1989), Marois and Croll (1992), and Kempf et al. (1997). The encapsulating membranes were removed from embryos (collected at days 2-5 after oviposition) by drawing the jelly mass and a small amount of seawater back and forth through an 18-gauge needle attached to a 3-ml disposable syringe. Hatched larvae (days 6-9) were collected from age-segregated cultures using glass Pasteur pipettes. Samples of embryos and larvae in seawater were placed into 1.5-ml Eppendorf centrifuge tubes with the addition of an approximately equal volume of 7.5% Mg[Cl.sub.2]. After centrifugation at about 400-600 X g for 5-10 s using a standard benchtop centrifuge, the supernatant was removed, and the tissues were fixed for 4-12 h, either in 4% paraformaldehyde in 0.1 mol [1.sup.-1] phosphate buffer (pH 7.4) at 4[degrees] C for eventual detection of 5-HT or FMRFamide-related peptides, or in methanol at -18[degrees] C for the detection of tyrosine hydroxylase (TH). After fixation, shells were decalcified in a saturated solution of EDTA in water for 10 min. The specimens were then washed several times in phosphate buffered saline (PBS; 50 mmol [1.sup.-1] [Na.sub.2]HP[O.sub.4] and 140 mmol [1.sup.-1] NaCl, pH 7.2) and then bathed overnight in blocking solution of 1% Triton X-100 and 1% bovine serum albumen in PBS. The specimens were next incubated for 2-3 d in one of the primary antibodies. The anti-5-HT and anti-FMRFamide sera were raised in rabbits and obtained from Diasorin, Inc. (now available through ImmunoStar, Hudson, WI). Both of these polyclonal antibodies were used at 1:500 dilution. The monoclonal anti-TH antibody was developed in mouse and also obtained from Diasorin. This latter antibody was used at 1:100 dilution.

Following incubation in a primary antibody, the larvae were given another three or four 1-h washes in PBS and then incubated for 12-24 h in goat anti-rabbit or sheep anti-mouse antibodies labeled with either FITC or rhodamine (Sigma Chemical Co., St. Louis, MO). Following another several washes in PBS, the specimens were mounted whole between glass cover slips in a solution of three parts glycerol to one part 0.1 mol [1.sup.-1] Tris buffer (pH 8.0) with the addition of 2% n-propyl gallate (Giloh and Sedat, 1982). Preparations were viewed and photographed on a Zeiss Axiophot microscope equipped with filter blocks with a 510-560-nm excitation and 590-nm longpass barrier filter for viewing rhodamine and a 450-490-nm and 515-565-nm bandpass barrier filter for viewing FITC.

Use of FITC- and rhodamine-labeled secondary antibodies yielded identical patterns of labeling. Staining patterns were also identical over the range of incubation and washing times used. Additional control experiments involved the use of procedures similar to those described above except for the replacement of the primary antiserum with either 1% normal mouse or rabbit serum or with the serum dilutant alone. No staining was observed in any of these control preparations. Finally, positive controls involved the co-processing of central ganglia, rhinophores, and tentacles from adult specimens of P. sibogae. Immunoreactivity in these organs is described elsewhere (Croll et al., 2001, 2003).

Photography

Histological preparations were photographed using Kodak TMAX 100 film. The negatives were digitally scanned, and the resultant images were then assembled into plates and labeled using Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA). Contrast and brightness of the images were adjusted to provide consistency within plates.

Results

General features of embryonic and larval development

The general features of the early development of Phestilla sibogae have been described previously (Bonar and Hadfield, 1974). These descriptions were confirmed and extended here and are summarized below (Fig. 1) to provide a context for subsequent descriptions of neural development. As might be expected from a study based upon the collection of eggs from a large population over an extended period of time, development was not tightly synchronized. Descriptions given below represent the majority of specimens found on any day. However, up to 20%-30% of specimens were found to be developmentally advanced or retarded by as much as one day.

On the second day after the eggs were laid (day 2; the earliest time systematically sampled in the present study), the embryos entered the early veliger stage and appeared to already be post-torsional. Both the velum and foot could be distinguished at the anterior end of the body, and a shell covered the posterior end. By day 3, the foot, velum, and shell had each enlarged, and additional features included statocysts, lightly pigmented eyes, and a small operculum. The most notable features of day 4-5 were elongation of the foot and slight increases in the size of the velum. The basic morphology of the larvae then remained largely unchanged from day 5 through day 8, with only a slight thickening of the foot observed near its anterior extreme and a slight decrease in the visceral mass as yolk stores were depleted. By day 9, the anterior end of the foot had bulged into a distinct propodium, and a space became noticeably larger over the visceral mass under the shell.

Serotonin (5-HT)-like immunoreactivity

The first 5-HT-containing cells appeared by day 2 (Fig. 2A). This pair of cells was located medial to the velar lobes, which were first developing at this time. Fibers projected medially across the midline to interconnect the cells. Other fibers projected laterally into the velar lobes. By day 3, these two cells were joined by three other apical neurons (Fig. 2B), which were often distinctly less immunoreactive than the first pair of 5-HT-like immunoreactive (lir) cells. The two most lateral of the later developing cells were slightly closer to the mouth than the first pair of apical neurons. The remaining single cell was positioned along the midline. All three of these cells were vase-shaped, with dendrites that projected to the epithelial surface. By this point, the apical organ also possessed a neuropilar region containing 5-HT-lir fibers beneath the somata. By day 4, the various 5-HT-containing fibers became more intensely stained as they projected into the neuropil and velum, with the elaboration of velar and apical innervation continuing through days 5 and 6 (Fig. 2C).

On day 7, 5-HT-lir was first detected in a bilaterally symmetrical pair of cells lying laterally to the apical organ in the region of the developing cerebral ganglion (Fig. 2D). By day 8, a second pair of 5-HT-lir somata was noted in the cerebral ganglia; by day 9, a total of three 5-HT-lir cells could be detected in each cerebral ganglion (Fig. 2E).

Days 7-9 also witnessed the first addition of 5-HT-lir cells to the pedal ganglia. On day 7, one pair of 5-HT-lir neurons could be observed in the pedal ganglia (Fig. 3A); on day 8, three pairs of 5-HT-lir pedal cells were observed (not shown); and by day 9, these cells were joined by a fourth pair (Fig. 3B). The development of the pedal ganglia coincided with the development of 5-HT-lir fibers that formed pronounced tracts running along each side of the foot (Fig. 3B, C). Immunoreactive somata, possessing what appeared to be projections to the periphery, were also located along these tracts (Fig. 3A, B). By day 8, the innervation to the foot became denser, and a rich network of 5-HT-lir fibers was present in the foot by day 9 (Fig. 3D).

The development of 5-HT-lir cells and fibers is summarized in Figure 4.

Tyrosine hydroxylase (TH)-like immunoreactivity

The first TH-lir cells also appeared by day 2 and were located lateral to the mouth. These cells remained the only TH-lir cells detected through day 3 (Fig. 5A, B), but by day 4 they were joined by a bilaterally symmetrical pair of cells in the velum as each lobe met the rest of the body near the eyes (Fig. 5C). By day 5, a similar pair of cells could be found ventrally in the velum at its junction with the body on each side near the mouth, as well as a single cell on the right side only, located slightly posterior and dorsal to the eye (Fig. 5D). Finally, also by day 5, a bilaterally symmetric pair of TH-lir cells could be observed in the region of the apical organ (Fig. 5E).

This pattern of nine cells remained unchanged on day 6, except by this time a fine immunoreactive fiber could occasionally be observed near the outer rim of the velum. On day 7, a few (5-10) TH-lir cells were observed in the foot. These cells roughly doubled in number by day 8, but by day 9 their numbers had risen until at least 40-60 cells could be counted. These cells were located throughout the foot but were especially concentrated in the propodium (Fig. 5F). By day 9, prominent fiber tracts could be seen running between the foot and the pedal ganglia. Fibers could also be observed along the outer rim of the velum (Fig. 5F), in the cerebral-pedal connective, and in the neuropil of the cerebral ganglia (not shown).

The development of TH-lir cells and fibers is summarized in Figure 6.

FMRFamide-like immunoreactivity

As with the other transmitter types, the first FMRFamide-lir cells were already present by day 2 (Fig. 7A, B). This bilaterally symmetrical pair of cells was located dorsally in the body and posterior to the velum. By day 3, these cells projected ventrally and anteriorly to brightly labeled regions of neuropil in the locations of the developing cerebropleural ganglia. By day 4, fibers projected across the midline to interconnect the neuropils on each side, and an additional neuropil could be detected in the region of the apical sensory organ (ASO). At this time an additional single cell also appeared on the midline, dorsal and posterior to the developing ASO (Fig. 7C). In addition to lying outside both the cerebral ganglia and the ASO, this cell was also distinct in its irregular, elongated shape and its bipolar projections dorsal to the eyes. By day 5, a single cell had been added near the apical neuropil, and immunoreactive fibers could be observed entering the velum (Fig. 7D). By day 6, an additional FMRFamide-lir somata had been added posterior to the cerebropleural neuropil on each side near the first immunoreactive neurons. At this point, fibers were observed projecting from the cerebropleural ganglion to neuropilar regions within the developing pedal ganglia (Fig. 7E-G). On the right side only, distinct projections could be observed innervating the larval kidney (Fig. 7F). By day 7, immunoreactive somata were detected near the tip of the foot, and fibers projected along each side of the foot toward the pedal ganglia (Fig. 7H). By day 9, a total of 5-7 immunoreactive somata could be observed near the tip of the foot (not shown).

The development of FMRFamide-lir cells and fibers is summarized in Figure 8.

Discussion

The present study is consistent with both the early appearance and the eventual extent of the larval nervous system noted in other gastropod molluscs. Cells containing 5-HT, catecholamines, and FMRFamide-like peptides were already present by the earliest veliger stages examined here, and it is likely that the first of these cells appeared during the trochophore stage of Phestilla sibogae, although this stage of development was not explored because preliminary studies indicated that the autofluorescent yolk obscurred visualization in these earliest embryos. Moreover, the timing and locations of many of the cells or cell populations noted here are similar to those reported in other species (reviewed in Croll and Dickinson, 2004). Specifically, for much of larval development, 5-HT-containing somata were confined to the apical sensory organ (ASO). Catecholamines were first detected around the mouth in early veliger stages but were also located in the velum and in numerous sensory-like cells of the foot in later stages. FMRFamide-like immunoreactivity was first located posterior to cerebral ganglia in an area that may correspond to pleural and parietal regions. Thus, it appears that the developmental program used by P. sibogae expresses many features conserved across the gastropods and that further studies on this species are likely to provide insights into general development within this taxon.

While the majority of cells described here lie outside of the boundaries of the ganglia that will eventually constitute the adult central nervous system (specifically the cerebropleural and pedal ganglia in nudibranchs like P. sibogae), this study nonetheless also permitted visualization of the developing ganglia as regions of neuropil that could be traced continuously throughout embryonic and larval development. For example, the cerebropleural ganglia were first detected as neuropilar regions rich in FMRFamide-like immunoreactivity at day 3. Similarly, the developing pedal ganglia was first detected at days 6 and 7 as neuropilar regions rich in FMRFamide- and 5HT-like immunoreactivity, respectively, but it was only on subsequent days that immunoreactive somata were detected within the ganglia. Thus, even though the present study is limited in its view of only a relatively few cells labeled for specific neurotransmitters, the development of these cells can be placed within a larger context of the developing central nervous system as a whole.

Serotonin (5-HT)

The most prominent 5-HT-lir cells during larval development were the five apical cells that were first described in P. sibogae by Kempf et al. (1997), who also correlated their anatomy with previous descriptions by Bonar (1978) of the ultrastructure of cells within the ASO. Thus the three vase-shaped cells, with slender apical dendrites, are referred to as the sensory (Type I) para-ampullary neurons, whereas the pair of round cells are referred to as the non-sensory (Type II) para-ampullary neurons. Kempf et al. (1997) also showed that nearly identical cells exist in several other nudibranch gastropods, and in fact, the conservation of these cells appears to be much more general. For instance, five cells with identical locations and morphologies have also been described within the ASO of the anaspidean Aplysia californica (Croll and Voronezhskaya, 1995; Marois and Carew, 1997b; Dickinson et al., 2000). Moreover, similar cells have been described in the ASOs of various caenogastropod species (Dickinson et al., 1999; Page and Parries, 2000; Dickinson and Croll, 2003), a palleogastropod (Page, 2002a), various polyplacophoran species (Voronezhskaya et al., 2002; Wanninger and Haszprunar, 2002), and even within bivalve (Croll et al., 1997; E. Voronezhskaya (Russian Acad. Sci), L. Nezlin (Russian Acad. Sci), J. Plummer (Dalhousie Univ.), unpubl. data) and scaphlopod (Wanninger and Haszprunar, 2003) molluscs, although the numbers of these apical serotonergic cells can vary (Page, 2002b). Interestingly, although these cells have now been noted widely within the molluscs, the exact timing of their appearances has been a subject of debate (Marois and Carew, 1997b; Page and Parries, 2000; Page, 2002a). The present study, however, provides evidence that the first serotonergic cells in P. sibogae are the round, non-sensory para-ampullary cells. Moreover, these cells appear to innervate the velum, and serotonergic innervation of the velum has previously been shown to involve the large preoral ciliary cells (Marois and Carew, 1997c) and thereby regulate swimming and feeding in the veliger larvae (Strathmann and Leise, 1979; Braubach et al., 2006).

The roles of the other 5-HT-containing apical cells are less clear. The structure of these cells suggests that they may have a sensory function, but their modality is not apparent from these anatomical studies alone. It has been shown, however, that 5-HT induces metamorphosis in another gastropod species (Couper and Leise, 1996) and that ablation of apical cells in P. sibogae renders larvae insensitive to a natural inducer of metamorphosis (Hadfield et al., 2000). Such studies implicate the vase-shaped sensory para-ampullary cells in the induction of metamorphosis, but the early appearance of these cells--long before the onset of metamorphic competence--argues for alternative or additional roles. Kuang et al. (2002) suggested that vase-shaped serotonergic cells in apical regions of a pond snail, Helisoma trivolvus, may detect oxygen levels in the environment. Alternatively, Voronezhskaya et al. (2004) suggested that monoaminergic neurons in apical regions of H. trivolvus and another pulmonate, Lymnaea stagnalis, might detect conspecific odors and use the sensory input to regulate rates of larval development. A similar role may therefore also exist for such cells in P. sibogae, although direct support for this possibility is currently lacking.

Just as the apical serotonergic neurons appear to be well conserved, the first 5-HT-containing cerebral cells may also be homologous across the several gastropod species examined to date. For example, the serotonergic cerebral cells described here and in other nudibranchs (Kempf et al., 1997) appear to be similar to those described in A. californica (Marois and Carew, 1997a, b). Moreover, details of timing, location, and eventual axonal projections of the cells in A. californica correspond with those described in the pulmonate Lymnaea stagnalis (Croll and Chiasson, 1989; Marois and Croll, 1992). These cells constitute a cluster of about five serotonergic cells that have been identified on the posterior dorsal surface of the cerebral ganglia of many other gastropods (Katz et al., 2001), including P. sibogae (Croll et al., 2001).

Similarly, the earliest serotonergic pedal cells appear to be comparable in time of appearance and possibly in locations to those in other heterogastropods (Marois and Croll, 1992; Marois and Carew, 1997b) and caenogastropods (Dickinson and Croll, 2003), and even to those in bivalves (Croll et al., 1997; E. Voronezhskaya, L. Nezlin, J. Plummer, unpubl. data). Early development of serotonergic pedal neurons is likely to reflect the need to regulate the activities of large numbers of ciliated cells found on the foot. Serotonergic regulation of ciliated cells by pedal neurons has been demonstrated in adult species of a variety of gastropod species (Audesirk et al., 1979; Syed and Winlow, 1989).

A unique finding here is of 5-HT-containing cells in the periphery near the tip of the foot. The morphology of cells with what appear to be dendrites projecting into the body wall suggests a sensory function, but the exact roles of the cells are unclear. It is also unknown whether such cells persist after metamorphosis, since 5-HT-containing cells appear to be rare or nonexistent in the periphery of adult specimens of gastropods (Sudlow et al., 1998), including P. sibogae (Croll et al., 2003), examined to date.

Tyrosine hydroxylase

Single large, catecholamine-containing cells on each side of the mouth appear to be common within the gastropods and bivalves examined to date (Croll et al., 1997, 1999; Dickinson et al., 1999, 2000; Dickinson and Croll, 2003). These cells generally first appear by the early veliger stage in other species, as they do in P. sibogae. In other species, however, these first two cells are later joined by several additional catecholamine-containing cells that generally have dendrites penetrating the overlying epithelium and therefore probably have sensory functions (see also Voronezhskaya et al., 1999). These additional cells, which have been hypothesized to monitor potential food quality and quantity in other species (Dickinson and Croll, 2003), were not observed in P. sibogae, which possesses sufficient yolk stores that it need not feed during larval life (Miller, 1993). However, it is also possible that such cells were simply not observed for technical reasons in the present study. The alcohol fixation needed to preserve TH-like immunoreactivity yielded less than optimal tissue preservation and fainter immunofluorescence than the aldehyde fixation employed for detection of 5-HT- and FMRFamide-like immunoreactivity. Small, faint cells and fibers (also see below) may therefore have been undetected. In addition, later developing cells may have been obscured by the large numbers of immunoreactive somata and fibers originating elsewhere in the larva.

Most gastropod and bivalve species examined to date also possess catecholamine-containing cells along the rim of the velum (Croll et al., 1997; Dickinson et al., 1999, 2000; Dickinson and Croll, 2003). Such cells appear to be interconnected by fibers that may also innervate the ciliated cells of the velum. P. sibogae, however, is exceptional in the paucity of these cells. All other species with free-living larval stages appear to have many such cells evenly spaced along the rim of the velum, whereas P. sibogae possesses only two such cells on each side--one positioned at each end of the velar rim. However, P. sibogae is the only nudibranch species in which catecholamine-containing cells have been described. The low numbers of these cells in the velum may therefore be a common feature of this taxon, which often has less elaborate vela than other gastropods (Buckland-Nicks et al., 2002). However, P. sibogae, which has a particularly short larval stage in which feeding is not obligatory (Miller, 1993), may possess even fewer velar elements than other species.

Like the catecholamine-containing cells around the mouth and the rim of the velum, the catecholamine-containing cells in the foot also appear to be general features of gastropod and bivalve larvae. In the present study, I demonstrated that a rapid increase in the numbers of these cells, particularly in the propodium, correlates with the onset of metamorphic competence at days 7-9 (Pires et al., 2000a). Pires et al. (2000a) also provided more detailed descriptions of these cells, which appear to lie subepithelially within the body wall of the foot. Moreover, previous studies demonstrated that the content of catecholamines (particularly dopamine) increased markedly in P. sibogae larvae at this time, and pharmacological experiments indicated that increased levels of catecholamines enhanced metamorphosis in this species whereas decreased levels inhibited it (Pires et al., 2000a). Similar evidence for catecholaminergic involvement in the induction of metamorphosis has been presented for other gastropod and bivalve species (Coon et al., 1985; Croll et al., 1997; Pires et al., 1997, 2000a, b; Pechenik et al., 2002). Together, these experiments suggest that the numerous catecholamine-containing cells may help to mediate the induction of metamorphosis. A sensory role is further suggested by the morphology of the cells with their apical dendrites (Pires et al., 2000a), and similar cells that form a metapodial ganglion in another heterobranch, On-chidoris bilamellata, have been reported to respond to chemical cues that trigger metamorphosis (Arkett et al., 1989). However, catecholamine-containing cells are also widespread in the body walls of many species of gastropods and bivalves (Smith et al., 1998; Croll et al., 1999, 2003; Croll, 2001, 2003), and the role of these cells does not appear to be solely devoted to the detection of induction-specific cues. The modality of the sensory cells, however, currently remains unclear, although mechanoreceptive functions have been suggested (Croll, 2003).

Although large numbers of catecholamine-containing cells seem to be similar to those previously described in other molluscan larvae, a few such cells appear to be novel. One such cell is represented by the unpaired, small soma located dorsal and posterior to the eye on the right side of larvae older than day 4. Unfortunately, the axon of this cell was undetected, so potential roles of the cell must remain speculative at present. The other novel catecholamine-containing cells were located in the ASO and appeared to be distinctly different in morphology and position from any of the 5-HT- or FMRFamide-lir cells described here. Catecholamine-containing cells seem to be rare within the ASO of gastropod and bivalve larvae (Croll et al., 1997; Dickinson et al., 1999, 2000; Dickinson and Croll, 2003), although one pair of such cells has been described in the apical region of the pond snail Lymnaea stagnalis (Voronezhskaya et al., 1999) and presumably represents the vestiges of the ASO in this pulmonate species (Croll, 2000). Clearly, a goal of future research must be to explain not only the similarities in the structure of the ASO in different species, but also the forces that cause some cell types to be found in certain species but not in others.

FMRFamide

FMRFamide-lir cells have been described in posterior regions of many molluscan larvae (Croll and Voronezhskaya, 1996; Dickinson et al., 1999, 2000; Croll, 2000; Dickinson and Croll, 2003; Croll and Dickinson, 2004). These cells normally first appear during the trochophore stage; by the early veliger stage, one or two cells are generally found on each side of the larvae near the developing parietal or pleural ganglia (or both) into which they project. Anterior projections of these cells eventually cross the midline in the developing cerebral commissure and also invade the developing pedal ganglia.

Unfortunately, visualization of any posterior cells in trochophore stages was obscured by the large autofluorescent yolk in P. sibogae. Nonetheless, the early developing FMRFamide-lir cells observed in the present study are consistent with other observations of FMRFamide-lir cells in gastropod larvae. At the earliest stages examined in P. sibogae, these cells already appeared to have formed extensive projections into the adjacent neuropil of the developing cerebropleural ganglia. Later, FMRFamide-lir fibers became apparent in the developing cerebral commissure and in the pedal ganglia, although some of this later immunoreactivity may have originated from additional neurons that appeared during subsequent larval development. Such similarities suggest that homology of the early FMRFamide-lir cells in other gastropods likely extends to the nudibranchs, despite significant differences in the development of the nervous system in these animals. Specifically, the adult nervous systems of nudibranchs manifest a high degree of cephalization with fusion of the cerebral and pleural ganglia, close proximity of the pedal ganglia with the cerebropleural ganglia, and an absence of major posterior ganglia. Indeed, in the present study I saw no evidence for the visceral loop of FMRFamide-lir fibers that has been described both before and after torsion in other species and suggests relatively anterior positioning of neural elements even at the earliest stages of development. Further studies into the early development of such cells may therefore provide insights into general trends in the organization of the nervous system that have long been noted to occur during the evolution of the molluscs.

Further studies on the FMRFamide cells in P. sibogae may also illuminate the eventual postmetamorphic fates of these cells, which remain unclear from studies of other species. In P. sibogae, the cells are easily visualized even in late stages of larval development. Furthermore, the distribution of FMRFamide-lir has now been studied extensively in both the central nervous system and regions of the periphery in postmetamorphic specimens of this species (Croll et al., 2001, 2003). Thus, future studies are now set to examine how the developing nervous system accommodates the dramatic changes in behavioral and physiological demands as such molluscs shift from pelagic, suspension-feeding larvae to crawling sea slugs that browse on hard coral in the benthos. To date, studies on the mechanisms by which nerve cells may be lost or gained or incorporated into new structures are almost entirely lacking in molluscs (but see a recent paper by Gifondorwa and Leise, 2006).

Although the most prominent FMRFamide-lir cells are located posterior to the developing central ganglia, other immunoreactive cells do appear during larval development. No midline FMRFamide has yet been described in dorsal positions of the head region of other molluscan larvae, but immunoreactive somata have been reported in both the apical organ and the foot of various larvae (Dickinson and Croll, 2003). The functions of these various cells, however, remain unknown.

Summary

The present study made it possible to describe in detail the locations and morphologies of cells containing different transmitters at comparable developmental stages within a single species. It appears that little overlap occurs between the different populations of neurons described here (although double-labeling experiments are needed to confirm the completeness of non-overlapping distributions), and thus the extensiveness of the larval nervous system is becoming apparent in the many cells described in this study. It is likely, however, that many more cells will be identified with the application of labels for other neurotransmitters (e.g., acetyl choline, [gamma]-aminobutyric acid, glutamate, histamine, and various neuropeptides) that have been suggested to be present in the adult nervous systems of other molluscs (Twarog, 1954; Osborne et al., 1972; Walker et al., 1975; Vitellaro-Zuccarello and De Biasi, 1988; Soinila et al., 1990; Croll and Van Minnen, 1992; Dale and Kandel, 1993; Willows et al., 1997; Chrachri and Williamson, 1998; Michel et al., 2000; Braubach and Croll, 2004).

In addition to demonstrating the extent of the larval nervous system, the present study also provides further insights into the structures of larval organs by elucidating their varied neural components. For example, the ASO in P. sibogae was first described by Bonar (1978) in terms of different cell types categorized on the basis of ultrastructure. More recently, Kempf et al. (1997) identified 5-HT as the major neurotransmitter located in a subset of five para-ampullary neurons within the ASO of P. sibogae and other nudibranchs. Here I identify three additional cells--two TH-lir cells and one FMRFamide-lir cell--with distinctly different shapes, sizes, positions, and timing of appearance within the ASO (Fig. 9). Such details may prove useful in comparisons of the ASO in different gastropods (Page and Parries, 2000; Page, 2002b) and shed light on the evolution of the ASO within the Metazoa (Lacalli, 1994; Nielsen, 1995; Hay-Schmidt, 2000).

Finally, in addition to permitting a more detailed understanding of the structure of the larval nervous system, the present study is one of the first to examine when cells containing several different neurotransmitters appear over much of the larval life of a nudibranch. Previous comparisons of neural elements in different gastropod species were hampered by uncertainty about whether differences represented phyletic changes in structure or developmental differences in the timing of appearance of specific cells. More comprehensive studies, such as the present research, permit resolution of this question. Neural elements identified in other molluscs can therefore be compared not only in terms of transmitter content, size, shape and position, but also in terms of ontogeny to suggest a basic plan for the larval nervous system, which appears to be well conserved over a wide phyletic range of molluscs with diverse larval life styles.

Acknowledgments

Sincere appreciation is extended to Michael Hadfield, his students Brian Nedved and Kim del Carmen, and the rest of the personnel at Kewalo Marine Lab, who provided invaluable help in the course of this work. This research was supported by Natural Sciences and Engineering Research Council of Canada grant #OPG38863 to Roger P. Croll and by Office of Naval Research grant # N00014-94-0524 to Michael G. Hadfield.

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ROGER P. CROLL

Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Received 17 March 2006; accepted 17 August 2006.

E-mail: roger.croll@dal.ca

Abbreviations: ASO, apical sensory organ; 5-HT, serotonin; lir, like immunoreactive; TH, tyrosine hydroxylase.
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