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Timing Is Everything: The Effects of Putative Dopamine Antagonists on Metamorphosis Vary With Larval Age and Experimental Duration in the Prosobranch Gastropod Crepidula fornicata

Biology Department, Tufts University, Medford, Massachusetts 02155

* To whom correspondence should be addressed. E-mail: jan.pechenik{at}tufts.edu

	Abstract

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The signal transduction pathway through which excess potassium ion stimulates the larvae of many marine invertebrates to metamorphose is incompletely understood. Recent evidence suggests that dopamine plays important roles in the metamorphic pathway of Crepidula fornicata. Therefore, we asked whether blocking dopamine receptors might prevent excess potassium ion from stimulating metamorphosis in this species. Surprisingly, the effects of the three putative dopamine antagonists tested (all at 10 µM) varied with exposure duration and the age of competent larvae. Chlorpromazine, a nonspecific dopamine antagonist known to have a number of other pharmacological effects, blocked the inductive action of excess potassium ion during the initial 5–8-h exposure periods in most assays, particularly for younger or smaller competent larvae. However, chlorpromazine in the absence of excess potassium ion also stimulated metamorphosis, particularly over the next 18 h, and worked faster on older competent larvae than on younger competent larvae. The specific D₁ antagonist R(+)-Sch-23309 had similar effects, blocking potassium-stimulated metamorphosis in short-term exposures and stimulating metamorphosis in longer exposures, particularly for older competent larvae. Although the specific D₂ antagonist spiperone (SPIP) blocked the inductive effects of excess potassium ion in only 1 of 6 assays during the first 6 h of exposure, it blocked metamorphosis in 2 of the assays during 24-h exposures. Our results indicate that dopamine receptors are involved in the pathway through which excess potassium ion stimulates metamorphosis in C. fornicata. In addition, the largely latent inductive effects of chlorpromazine, an inhibitor of nitric oxide synthase, suggest that endogenous nitric oxide may play a natural role in inhibiting metamorphosis in this species. Overall, our results would then suggest that exposing larvae of C. fornicata to excess K⁺ leads to a shutdown of nitric oxide synthesis via a dopaminergic pathway, a pathway that can be blocked by some dopamine antagonists. Alternatively, chlorpromazine might eventually be stimulating metamorphosis by elevating endogenous cyclic nucleotide (e.g., cAMP) concentrations, again acting downstream from the steps acted on directly by excess K⁺.

	Introduction

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As with most other marine gastropods, Crepidula fornicata has an obligate planktonic larval stage. After swimming and feeding in the plankton for at least several weeks, the veliger larvae become competent to metamorphose (Pechenik, 1990); that is, they become capable of metamorphosing in response to specific external cues such as adult pheromone and microbial films (Pechenik, 1980; Pechenik and Heyman, 1987; McGee and Targett, 1989; Pechenik and Gee, 1993). In the absence of such external cues, the larval form can be maintained for at least an additional 10 days (Pechenik and Lima, 1984; Zimmerman and Pechenik, 1991). Eventually the larvae metamorphose "spontaneously" in frequently cleaned glassware, in the apparent absence of external cues (Pechenik, 1984; Pechenik and Lima, 1984; Pechenik et al., 1996a).

The signal transduction pathway that leads from perception of the external cue to metamorphosis is incompletely understood in this species, and indeed in any marine invertebrate species (Todd et al., 1991; Pechenik et al., 1995; Clare, 1996; Woollacott and Hadfield, 1996; Pechenik and Qian, 1998; Carpizo-Ituarte and Hadfield, 1998; Holm et al., 1998; Leise and Hadfield, 2000; Leise et al., 2001). Recently, Pires et al. (2000b) found that artificially depleting the concentration of endogenous dopamine and its precursor L-DOPA to about 50% of initial concentrations inhibited larvae of Crepidula fornicata from metamorphosing in response to adult-conditioned seawater, suggesting that catecholamines play an important role in regulating metamorphosis in this species. Similarly, dopamine and other catecholamines seem to function in the metamorphic pathway of the opisthobranch gastropod Phestilla sibogae (Pires et al., 2000a). In those experiments with P. sibogae, increasing the endogenous dopamine concentration of competent larvae by 20–50 times significantly increased the sensitivity of larvae to natural inducer (an extract of the coral Porites compressa).

In this study we sought direct evidence for the involvement of dopamine receptors in the metamorphic pathway of Crepidula fornicata. Such studies first require the ability to reliably identify when larvae become physiologically competent to metamorphose, and the ability to subsequently induce all competent larvae to metamorphose. Although competent larvae of C. fornicata can be induced to metamorphose using seawater conditioned by adults (Pechenik, 1980; Pechenik and Heyman, 1987; McGee and Targett, 1989), the results are quite variable from one experiment to the next. When larvae fail to respond to the adult-conditioned seawater, is it because the larvae were not yet competent to respond, or is it because the adults simply did not produce enough cue to induce a response? We cannot distinguish between these possibilities because the adult cue is still only partially defined (McGee and Targett, 1989). Exposure concentrations cannot be standardized in such a situation, and the extent of physiological competence within larval populations cannot be accurately quantified (Pechenik and Heyman, 1987).

However, metamorphosis can be reliably induced in C. fornicata artificially, by incubating larvae for 5–6 h in seawater in which the concentration of K⁺ has been raised by 15–20 mM (Pechenik and Heyman, 1987; Pechenik and Gee, 1993; Pires et al., 2000b). The larvae become competent to respond to excess K⁺ only about 12–24 h before they become competent to respond to natural inducer, following 2–3 weeks of development as pre-competent larvae (Pechenik and Gee, 1993). This is not the case for all species: the larvae of at least some other marine invertebrate species become responsive to excess K⁺ well after they become responsive to natural cues (Pechenik et al., 1995; Pechenik and Qian, 1998). Exposing larvae to excess K⁺ provides a reliable way to monitor the onset of metamorphic competence within laboratory populations of C. fornicata.

Moreover, metamorphosis in C. fornicata—signaled by loss of the larval swimming organ, the velum—is comparably rapid in response to natural inducer and excess K⁺, typically requiring less than 6 h in both cases (Pechenik and Gee, 1993). Finally, triggering metamorphosis of C. fornicata with excess K⁺ has no detrimental effect on rates of juvenile growth, feeding, or respiration (Eyster and Pechenik, 1988).

In this study, we used excess K⁺ as a convenient tool to explore aspects of signal transduction in the metamorphic pathway of C. fornicata. In particular, we examined the ability of several specific and nonspecific putative dopamine antagonists to block the ability of excess K⁺ to stimulate metamorphosis. Although dopamine receptor types have not yet been characterized in this species, dopamine is known to play several important roles in molluscan physiology, and its various effects can be blocked by a variety of mammalian dopamine receptor antagonists in a number of other gastropod species (e.g., Juel, 1981; Swann et al., 1982; Kim and Woodruff, 1995; Emaduddin and Takeuchi, 1996; Green et al., 1996; Spencer et al., 1996).

Our study differs from most previous studies exploring the effects of neuroactive and pharmacological agents on gastropod metamorphosis in that instead of using larvae of the same age in each experiment (e.g., Bryan and Qian, 1998; Holm et al., 1998; Carpizo-Ituarte and Hadfield, 1998) or subsampling larvae at intervals only from pre-competence to the onset of competence (e.g., Pechenik and Qian, 1998; Pires et al., 2000a), we subsampled from the same batches of competent larvae over time. In so doing, we encountered the surprising and potentially revealing result that the effects of particular treatments changed dramatically as competent larvae aged. Moreover, instead of ending our experiments at 6 h, we continued them for an additional 18 h, encountering the equally remarkable result that some treatments had the opposite effect at 24 h as they had at 6 h. We believe this to be the first published demonstration of such phenomena for larval invertebrates. The results suggest new hypotheses about how metamorphosis is controlled in this species.

	Materials and Methods

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Obtaining competent larvae
Four experiments were conducted, each using larvae released from a different female. Several stacks of adults were collected from Nahant, Massachusetts, at low tide and held in the laboratory on a diet of the naked flagellate Dunaliella tertiolecta (clone DUN) for 2–6 days until larvae were released. For each of our four main experiments, about 3000 larvae were then reared in 4-1 glass jars in 0.45-µm filtered seawater from Nahant (salinity about 30) on a diet of the naked flagellate Isochrysis galbana (clone T-ISO). This diet supports rapid growth of these larvae with low mortality (e.g., Pechenik, 1984; Pechenik and Gee, 1993). The water and phytoplankton suspensions were changed every other day, and the glassware was cleaned between water changes. After 10–12 days, larvae were subsampled periodically (three replicates of 10 larvae each) and tested for metamorphic competence by raising the K⁺ concentration of seawater by 20 mM for 6 h (Pechenik and Heyman, 1987; Pechenik and Gee, 1993). Assays in the first three experiments were initiated once at least 80% of the tested larvae were competent to metamorphose, as described below. In the last experiment (Experiment IV) we initiated our tests when only about 30% of larvae were competent to metamorphose. This was done deliberately, so as to initiate assays when most larvae in the culture were just beginning to acquire metamorphic competence.

Testing the effects of putative dopamine antagonists and a nitric oxide synthase inhibitor
Our experiments focused on the nonspecific (Green et al., 1996) dopamine antagonist chlorpromazine; chlorpromazine has a number of other documented actions, including the inhibition of nitric oxide synthase (see Discussion). A smaller number of studies also included the selective D₂ antagonist spiperone (SPIP) and the D₁ antagonist R+ (R(+)- Sch-23309). Because nitric oxide may be a natural inhibitor of metamorphosis in another gastropod (Ilyanassa obsoleta; Leise et al., 2001), we also tested the effects of the nitric oxide synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester) in two experiments.

Chlorpromazine was initially tested at concentrations of 5, 10, 20, and 40 µM in seawater. For subsequent experiments the chemical was applied only at 10 µM, because that concentration produced a maximal response with minimal larval mortality (see below). The two other dopamine antagonists were tested only at 10 µM, and L-NAME was tested at 10 and 20 µM. The concentrations of chlorpromazine, SPIP, and R(+)-Sch-23309 that we used are similar to those used in the studies of Croll et al. (1997) with Lymnaea stagnalis and of Green et al. (1996) with Helisoma trivolvis. Micromolar concentrations of L-NAME (injected) stimulated metamorphosis in Ilyanassa obsoleta (Leise et al., 2001).

Exposure protocol
In all experiments, larvae of C. fornicata were exposed to test solutions in sets of four replicates with 8–12 larvae (usually 10) per replicate. When subsampling larvae for different assays within an experiment, we selected the largest larvae, discarding any that were unusually large. Filtered seawater served as the negative control. A positive control, to assess the percentage of larvae that were competent to metamorphosis on the day of each experiment, consisted of seawater whose K⁺ concentration had been elevated by 20 mM (Pechenik and Heyman, 1987; Pechenik and Gee, 1993). Other larvae were exposed either to the test solutions alone (chlorpromazine, SPIP, R+) or to the test solutions in the presence of excess K⁺ (20 mM). In this way, the ability of the tested substances to block the effects of excess K⁺ on metamorphosis was assessed.

The percentage of larvae metamorphosing in each solution was assessed after 6–8 h of exposure (usually 6 h). Percent metamorphosis was also assessed after 24 h. Mean shell lengths of larvae used in each experiment were determined by subsampling at least 12 larvae and measuring them at 50x; those larvae were not used in the experiments, to guard against the possibility that the handling might stimulate metamorphosis. Metamorphosis was signaled by loss of the ciliated larval swimming organ, the velum, as assessed with a dissecting microscope at 50x.

Within each large (numbered) experiment, the exposures were repeated up to 5 times by subsampling from the same batch of larvae over 13–15 days, to determine whether the responses changed as the competent larvae aged. Individual larvae were tested only once. Experimental details, including larval age and mean shell length at each exposure, are given in Table 1.

Data analysis
Percentage data were arcsine transformed before analysis (Sokal and Rohlf, 1981). Mean responses were compared by one-way analysis of variance (ANOVA) unless variances did not pass Bartlett’s test of homogeneity, in which case means were compared using nonparametric ANOVA (Kruskal-Wallis KW statistic). When significant differences among means were found, treatment means were compared against control means using Bonferroni’s method for multiple comparisons.

Mean shell lengths of larvae used in experiments were compared using one-way ANOVA on untransformed data, followed by Bonferroni’s method for multiple comparisons (sample means against mean shell length at the first assay within an experiment).

All analyses were conducted using the programs Prism or Instat (both from GraphPad, Inc.).

	Results

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The effects of chlorpromazine on larvae of Crepidula fornicata were consistent among the four experiments (each using larvae from a different female), but varied in interesting and unexpected ways with exposure time and larval age within experiments. With only one exception, at least 50% of larvae metamorphosed within 5–6 h when subjected to 20 mM excess K⁺ (Fig. 1a). The lower initial response in Experiment IV was an intended outcome of our experimental design, as we wished to begin testing the responses of larvae that were just becoming competent to metamorphose; by the third assay in that experiment, over 90% of larvae were competent to metamorphose. In one experiment (Experiment IIIa), larvae took 2–3 h longer to respond to the excess K⁺ than in previous studies, so that initial responses were recorded at 8 h rather than at 6 h. Fewer than 3% of larvae metamorphosed during the first 5–8 h in seawater controls (Fig. 1a); indeed, fewer than 10% of larvae had metamorphosed in seawater controls by the end of 24 h (Fig. 1b). Significantly more larvae (P < 0.01) metamorphosed in response to excess K+ than in control seawater in all experiments (one-way ANOVA followed by Bonferroni comparisons between means).

View larger version (52K): [in this window] [in a new window]   Figure 1. The influence of larval age and exposure duration on the effects that chlorpromazine has on metamorphosis in larvae of Crepidula fornicata. Excess K+ was used as a convenient and reliable method for inducing metamorphosis. (a) Data for the first 6–8 h of exposure. (b) Data for the first 24 h of exposure. Larvae were obtained from a different female for each of the four experiments. Each bar represents the mean response (+ 1 SD) of larvae in 4 replicates, 10–12 larvae per replicate. "1" indicates a significant difference in the means of chlorpromazine and control outcomes (P < 0.05); i.e., that chlorpromazine stimulated metamorphosis significantly above control levels. "2" indicates a significant difference in the means of excess K+ and (chlorpromazine + excess K+) outcomes; i.e., that chlorpromazine significantly blocked the stimulatory effects of excess K+. ND = No Data. The numbers in parentheses below the x-axis give the ages of the larvae when tested, in days. The dates of each assay, along with mean shell lengths of the larvae tested, are given in Table 1.

As larvae aged within Experiments I and IV, however, chlorpromazine itself stimulated substantial metamorphosis within the same 5–8-h exposure periods, and at least in some assays, apparently became less effective in blocking stimulation by excess K⁺. For the larvae used in Experiment I, for example, only about 10% of the larvae that were subsampled on 17 May 2000 (Experiment Ia) metamorphosed in 10 µM chlorpromazine within the first 6 h of exposure. But when larvae from the same batch were subsampled and tested 13 days later (Experiment If), 35% of larvae tested at the same chlorpromazine concentration metamorphosed in the same amount of time, and chlorpromazine did not inhibit the action of excess K⁺ at all (Fig. 1a). In some assays, however, chlorpromazine stimulated metamorphosis of older larvae while simultaneously suppressing the inductive effects of excess K⁺ (see Experiment III, for example, Fig. 1a).

Furthermore, over the subsequent 18 h or so that larvae were exposed, chlorpromazine alone stimulated metamorphosis in all experiments, even for the youngest and smallest larvae tested (see Table 1), relative to the incidence of metamorphosis occurring in control seawater (Fig. 1b, indicated by the numeral "1" above the relevant bars). In fact, about as many larvae metamorphosed in response to chlorpromazine by the end of 12 h as had metamorphosed in response to excess K⁺ in the first 6–7 h (Fig. 2). By the end of 24 h chlorpromazine had stimulated at least 80% of larvae to metamorphose in 11 of 12 assays, even when simultaneously blocking stimulation by excess K⁺ as in Experiments IIIb and IIIc (Fig. 1b). In addition, chlorpromazine in 24-h assays generally failed to block the stimulatory effects of excess K⁺ significantly.

View larger version (16K): [in this window] [in a new window]   Figure 2. The time-course of larval responsiveness to chlorpromazine and excess K+. Each point represents the mean response of larvae in four replicates, 10 larvae per replicate.

View larger version (43K): [in this window] [in a new window]   Figure 3. The influence of larval age on the effectiveness of the D1 antagonist R+ in suppressing metamorphosis and in inducing Crepidula fornicata larvae to metamorphose within the first 5–6 h (a) and first 24 h (b) of exposure. Each bar represents the mean response (+ 1 SD) of larvae in four replicates, 10–12 larvae per replicate. "1" indicates a significant difference in the means of R+ and control outcomes (P < 0.05); i.e., that R+ stimulated metamorphosis significantly above control levels. "2" indicates a significant difference in the means of excess K+ and (R+ plus excess K+) outcomes; i.e., that R+ significantly blocked the stimulatory effects of excess K+. The numbers in parentheses below the x-axis give the ages of the larvae when tested, in days. The dates of each experiment are given in Table 1.

View larger version (34K): [in this window] [in a new window]   Figure 4. The influence of larval age on the effectiveness of the specific D2 antagonist SPIP (spiperone) in suppressing metamorphosis and in inducing Crepidula fornicata larvae to metamorphose within the first 6–8 h (a) and first 24 h (b) of exposure. Each bar represents the mean response (+ 1 SD) of larvae in four replicates, 10–12 larvae per replicate. "1" indicates a significant difference in the means of SPIP and control outcomes (P < 0.05). "2" indicates a significant difference in the means of excess K+ and (SPIP + excess K+) outcomes. The numbers in parentheses below the x-axis give the ages of the larvae when tested, in days. The dates of each experiment are given in Table 1.

	Discussion

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It is always difficult to fully interpret the results of studies in which intact, microscopic animals are immersed in baths of pharmacological agents (Pawlik, 1990). In our studies, problems include not knowing exactly where agents act, or when (or whether) they reach target tissues; the possibility that particular agents will have multiple (and perhaps simultaneous) effects within individual organisms; and uncertainty as to whether substances will operate in the target animal in the same way as they have been demonstrated to operate in the model organism or tissue preparations in which effects have been well-defined. However, the results of these experiments seem clear and convincing, even though our interpretation of those results may be controversial or incomplete. Chlorpromazine, a nonspecific dopamine antagonist, blocked the stimulatory action of excess K⁺ clearly and dramatically for competent larvae of Crepidula fornicata during the first 6–8 h of exposure, particularly for the younger competent larvae within an experiment, and the specific D₁ antagonist R+ had a similar effect (indicated by the numeral "2" above the relevant bars of Figs. 1aand 3a). The results suggest that D₁ receptors are involved in the signal transduction pathway stimulated by excess K⁺ in this species. Because natural cues probably act directly on external sensory receptors at the beginning of the metamorphic pathway (e.g., Baloun and Morse, 1984; Hadfield et al., 2000), upstream from wherever the excess K⁺ is acting, we predict that—in short-term assays—chlorpromazine and R+ will also be found to block metamorphosis stimulated by natural cue in this species.

On the other hand, the specific D₂ antagonist SPIP blocked the inductive effect of excess K⁺ in only 1 of 6 short-term assays, and even then by only about 30% (Experiment IIIa, Fig. 4a). Possibly the SPIP was simply not reaching the active site within the larvae quickly enough to block the stimulatory effect of excess K⁺ during 6-h exposures. If so, a 1–2-h pre-exposure to SPIP might have been more effective at inhibiting metamorphosis in response to an increase in ambient K⁺ concentration. This seems likely: although SPIP blocked the stimulatory effect of excess K⁺ in only one experiment during 5–6-h exposures (Fig. 4a), it blocked the effect of excess K⁺ significantly in two experiments by the end of 24-h exposures (Fig. 4b). Because SPIP is more lipophilic than the other two chemicals that we tested (the other two chemicals are water soluble, but SPIP is not), it should cross cell membranes more readily and have easier access to sites that are not so readily accessible to R+ or chlorpromazine; its limited ability to block the effects of excess K⁺ in our studies suggests that it is acting at sites much farther downstream in the signal transduction pathway than the sites acted on by the other two chemicals. That the stimulatory effect of SPIP was slow to develop in these experiments is consistent with this suggestion. Our results with SPIP therefore suggest that D₂ receptors are also involved in the signal transduction pathway of C. fornicata. Overall, our data indicate that dopamine receptors may operate at several steps in the signal transduction pathway and lend support to the recent indications (Pires et al., 2000b) that dopamine and perhaps other catecholamines play important roles in regulating metamorphosis in this species.

On the other hand, Pires et al. (2000b) found that artificially depleting the concentration of endogenous dopamine and its precursor L-DOPA to about 50% of initial concentrations in larvae of C. fornicata did not inhibit them from metamorphosing in response to excess K⁺, even though it did inhibit the response to adult-conditioned seawater. We offer two explanations for this apparent discrepancy. One possibility is that neuronal concentrations of catecholamines may have been greatly reduced in some tissues but not significantly reduced in others; dopamine and L-DOPA concentrations were determined by Pires et al. (2000b) from pools of 30–40 whole larvae, so that it was not possible to tell whether dopamine and L-DOPA concentrations were reduced to the same extent in all tissues. Another possibility is that adult-conditioned seawater may be a weaker stimulus than excess K⁺, causing a weaker depolarization and therefore less dopamine to be released. If so, excess K⁺—but not adult-conditioned seawater—might still be able to cause the release of sufficient dopamine to induce metamorphosis even when cellular dopamine concentrations are artificially reduced.

It is also clear from our data that chlorpromazine, while acting to block the stimulatory action of excess K⁺ on metamorphosis, was itself a stimulus to metamorphosis, particularly for older competent larvae and in the longer (24-h) assays. Chlorpromazine has several additional effects in other model systems that may explain this aspect of our data. Of particular interest is that, in addition to its role as a dopamine D₂ antagonist, chlorpromazine inhibits nitric oxide synthase (NOS). Froggett and Leise (1999) present evidence that endogenous NO inhibits metamorphosis in the prosobranch gastropod Ilyanassa obsoleta. They found that although serotonin (5-HT) injections stimulated larvae of that species to metamorphose, the stimulatory effect of serotonin was blocked by bath application of the NO donors SIN-1 and SNAP (Froggett and Leise, 1999; Leise et al., 2001). Also, they were able to induce metamorphosis by artificially reducing endogenous NO concentrations using the NOS inhibitor L-NAME (Froggett and Leise, 1999; Leise et al., 2001). Similarly, endogenous NO apparently represses metamorphosis in two ascidian species (Bishop et al., 2001) and the sea urchin Lytechinus pictus (Bishop and Brandhorst, 2001). This could explain the latent stimulatory effect of chlorpromazine on larvae in our experiments if metamorphosis in C. fornicata is also under insect-like inhibitory control (Pechenik and Qian, 1998), and assuming that chlorpromazine takes longer than K⁺ to reach its target. It cannot, however, explain the latent stimulatory effect of R+, unless that chemical also has (unexplored) multiple effects in this system. Chlorpromazine can act as a histamine H₁ antagonist in some systems (Martinez and Coleman, 1990; Oken, 1995) and may also elevate intracellular concentrations of cyclic AMP or cGMP (by inhibiting calmodulin-stimulated cyclic nucleotide phosphodiesterase; Alfonso et al., 1995), offering further possibilities for future exploration. Elevated concentrations of cAMP play roles in the signal transduction pathway of insect (Smith, 1995; Gilbert et al., 1997) and barnacle metamorphosis (Balanus amphitrite: Clare, 1996; Yamamoto et al., 1998), although apparently not in the metamorphic pathway of the polychaete Hydroides elegans (Holm et al., 1998). Finally, chlorpromazine depletes serotonin in the central nervous system of Lymnaea stagnalis, but only after exposures of much greater duration than those used in the present study (Croll et al., 1997). Thus it is also possible, though less likely, that chlorpromazine in our experiments stimulated metamorphosis through an effect on serotonin concentration.

Competent larvae of C. fornicata become sensitive to excess K⁺ about 12–24 h sooner than they become responsive to adult-conditioned seawater, suggesting that excess K⁺ acts at a point (or points) somewhat farther downstream from where the natural cue acts (Pechenik and Gee, 1993; see also Hadfield et al., 2000). We cannot exclude the possibility that the excess K⁺ may also depolarize the same sensory receptor or receptors eventually acted upon by the natural chemical stimulus; the sensory neurons might become sensitive to excess K⁺ before the number of receptors on those neurons is sufficient to allow depolarization by natural chemical stimuli. Regardless of exactly where and at how many points excess K⁺ acts, our working hypothesis is that exposing larvae of C. fornicata to excess K⁺ leads to a shutdown of NO synthesis via a dopaminergic pathway—a pathway that can be blocked by some dopamine antagonists. Our results also suggest that chlorpromazine has an additional effect deeper in the metamorphic pathway of this species, downstream from the dopamine receptor block, possibly shutting down NO synthesis directly. The failure of the NOS inhibitor L-NAME to stimulate metamorphosis in our experiments could be an artifact of bath application; Froggett and Leise (1999) stimulated metamorphosis in Ilyanassa obsoleta through injection rather than bath application.

Alternatively, chlorpromazine might instead (or in addition) be stimulating metamorphosis in this species by elevating concentrations of calmodulin-dependent cyclic nucleotide phosphodiesterase. Either of these secondary roles could explain why chlorpromazine can act both to inhibit the stimulatory effects of excess K⁺ and to stimulate metamorphosis on its own.

It is also clear that the responsiveness of C. fornicata larvae to chlorpromazine and R+ changed over time in our experiments, probably reflecting age-related changes in larval physiology. Such age-related changes in response were seen even in Experiment I, in which the mean shell length of larvae tested in the different assays did not increase significantly over time (Table 1, Fig. 1). Previous studies with this species indicate that neither absolute size nor absolute age are good indicators of physiological state (Pechenik and Heyman, 1987; Pechenik et al., 1996b). It is also possible that the different results of assays within an experiment reflect physiological differences between faster and slower-growing larvae, since for each assay we deliberately selected the largest individuals available within a culture (although avoiding any unusually large larvae). Assuming that the different responses are in fact age-related, our results may have some bearing on the phenomenon of "spontaneous metamorphosis" (Crisp, 1974; Pechenik, 1984, 1990). If metamorphosis of competent larvae is prevented, in the absence of an appropriate external cue, by the presence of an endogenous inhibitor such as NO, then spontaneous metamorphosis may reflect an eventual end to the endogenous production of that inhibitor (Chia, 1978; Pechenik and Qian, 1998). A gradual decline in inhibitor titer might explain the age-related effects of chlorpromazine on metamorphosis documented in this study: chlorpromazine, acting as an inhibitor of NOS, would have a more rapid effect on metamorphosis of older competent larvae, as seen in the present study, if NO titers were declining naturally as the larvae aged. More detailed work on the effects of NO donors and NOS inhibitors in competent larvae of C. fornicata, following the protocols of Froggett and Leise (1999), could be productive, along with additional studies on the potential role of cAMP (and cGMP) in the signal transduction pathway.

It is worth pointing out that the complexity of our results arises from the apparently novel way that we conducted these experiments. If we had tested larvae at one age—for example when at least 60% of larvae subsampled from a culture were first competent to metamorphose—and if we had monitored our assays only at 24 h, our results would probably have been more straightforward. In general, we would have found that the putative dopamine antagonists that we tested did not block the effects of excess K⁺, and we would have failed to find substantial support for the involvement of dopamine receptors in the metamorphic pathway, or at least that portion of the pathway activated by excess K⁺. On the other hand, had we sampled only at 5–6 h, our results would have been far more erratic and difficult to interpret, both in the ability of the pharmacological agents to block the effects of excess K⁺ and in their ability to stimulate metamorphosis directly (Figs. 1a, 3a). Only by monitoring changes in the responses of competent larvae subsampled from individual cultures over time were we able to determine that the ability of chlorpromazine and R+ to block the stimulatory effects of excess K⁺ diminished as competent larvae aged, and that the stimulatory effects of the pharmacological agents themselves increased as the larvae aged and as experimental duration was prolonged. Perhaps the most interesting contribution of the present study is its demonstration that the effects of pharmacological agents on metamorphosis can change dramatically as competent larvae age, and as experimental duration is changed: timing is everything. It might be worth revisiting other marine invertebrate models in this light.

	Acknowledgments

 
We thank Drs. Tony Pires, David Macmillan, and Charles Derby, and two anonymous reviewers, for their detailed comments and helpful suggestions on a draft of the manuscript.

	Footnotes

 
Received 13 August 2001; accepted 11 January 2002.

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