Gaseous nitric oxide (NO) is produced through the action of the enzyme nitric oxide synthase (NOS) and acts as a neurotransmitter (Jacklet and Gruhn, 1994b;Elphick et al., 1995a;Jacklet, 1995) in the nervous systems of adult gastropod molluscs. By comparison, little or no information appears to exist about the ontogeny of molluscan NOS-containing neurons. NADPH-diaphorase (NADPHd) has been determined biochemically and histochemically to colocalize with NOS immunoreactivity in neurons; NOS is an isoform of NADPHd (Dawson et al., 1991;Hope et al., 1991). We used NADPHd histochemistry to map the distribution of NOS activity in the nervous systems of larvae, including metamorphosing individuals, and juveniles of the marine snail Ilyanassa obsoleta. Several ganglionic neuropils displayed reaction product throughout development. The most intense NADPHd staining occurred in the neuropil of the apical ganglion, a specialized larval structure. Intermediate staining levels occurred in neuropils of the cerebral, pedal, and pleural ganglia. Larval buccal and intestinal ganglia showed little reaction product, with slight increases arising in metamorphically competent larvae. NADPHd activity conspicuously decreased in the central nervous systems of metamorphosing larvae. The osphradial ganglion, which was present in young larvae, showed only weak NADPHd activity. Our results provide evidence for the existence of a nitrergic signalling system in molluscan larvae and juveniles.

We determined that the larval nervous system of Ilyanassa obsoleta contains paired cerebral, pleural, pedal, buccal and intestinal ganglia and unpaired apical, osphradial, and visceral ganglia. We used a modified form of NADPH diaphorase histochemistry to compare the neuroanatomy of precompetent (including specimens 6, 8, and 12 days after hatching), competent, and metamorphosing larvae with postmetamorphic juveniles. This method highlighted ganglionic neuropils and allowed us to identify individual ganglia at various stages of development, thereby laying a foundation for concurrent histochemical studies. The first ganglia to form were the unpaired apical and osphradial ganglia and the paired cerebral and pedal ganglia. In larvae 6 days after hatching, the neuropil had already appeared in the apical and osphradial ganglia. Neuropil began to be apparent in the cerebral and pedal ganglia 2 days later. At that time, the pleural and buccal ganglia were identifiable and adjacent to the posterior edge of the cerebral ganglia. The ganglia of the visceral loop were concurrently recognizable, although the supraintestinal ganglion developed slightly earlier than the subintestinal and visceral ganglia. By 12 days after hatching all of the major adult ganglia were discernible. The apical ganglion was retained by newly metamorphosed juveniles, but not by juveniles 2 days later. After metamorphosis was complete, the central nervous system (CNS) was consolidated into its juvenile form with ipsilateral cerebral and pleural ganglia being partially fused. The metamorphic translocation of ganglia, which included a caudal relocation of the cerebrals and the migration of the buccals from above the esophagus to a position below it, correlated with the movement of the proboscis to the dorsal part of the head.