This investigation provides a light and electron microscopic examination of the development of serotonin-like immunoreactivity and structure of the apical sensory organ (ASO) in embryos and/or larvae of four nudibranch species: Berghia verrucicornis, Phestilla sibogae, Melibe leonina, and Tritonia diomedea. Serotonin-like immunoreactivity is first expressed in somata, dendrites, and axons of a group of five distinct neurons within the ASO. These neurons extend axons into an apical neuropil, a structure that is situated centrally and immediately dorsal to the cerebral commissure. Three of these neurons possess sensory dendrites that extend through the pretrochal epithelium, each supporting two cilia at their distal ends. Later development of serotonin-like immunoreactivity includes 1) axons from the apical neuropil that extend into each of the velar lobes; 2) neuron perikarya in the cerebral and pedal ganglia; 3) axons that extend through the cerebral commissure, cerebral-pedal connectives, pedal commissure, and possibly the visceral loop connective; and 4) axons extending from each pedal ganglion into the larval foot. Ultrastructurally, the ASO can be seen to be composed of three lobes and an apical neuropil that is separately delineated from the cerebral commissure. Four cell types are present within the ASO: ciliary tuft cells, type I and type II parampullary neurons, and ampullary neurons. Immunofluorescence and 3,3' diaminobenzidine tetrahydrochloride (DAB) labeling verify that the serotonergic neurons of the ASO are type I and type II parampullary neurons. The ampullary and type I parampullary neurons possess dendrites that extend through the pretrochal epithelium. These dendrites are partitioned into three bundles, one on either side of the ciliary tuft cells and a third bundle penetrating the pretrochal epithelium centrally between the ciliary tuft cells. One serotonergic type I parampullary neuron is associated with each of these bundles. Two ampullary neurons are associated with each of the lateral dendritic bundles, while the central bundle includes only one. Ultrastructural analyses of serotonergic axonal innervation arising from the ASO agree with those determined from fluorescently labeled material. The structure of the ASO and its associated serotonergic axons suggest that the serotonergic component of this structure senses environmental stimuli affecting velar function, possibly the contractility of muscle fibers in the velar lobes. Similarities and differences among the ASOs of embryos and larvae from various invertebrate phyla may provide useful data that will assist in the reconstruction of phylogenetic relationships.

The "symbiosome membrane" as defined by Roth et al. (1988) is a single, host-derived membrane that surrounds an endosymbiotic organism, separating it from the cytoplasm of the host cell. However, in the case of cnidarian-dinoflagellate endosymbioses, clear identification of the symbiosome membrane is complicated by the fact that each algal symbiont is surrounded by multiple layers of apparent membrane. The origin and molecular nature of these membranes has been the subject of considerable debate in the literature. Here we report the development of host-specific (G12) and symbiont-specific (PC3) monoclonal antibodies that allow separation of the host and symbiont components of these multiple membranes. Using immunocytochemistry at both the light and the electron microscopic level, we present data supporting the conclusion that the definitive symbiosome membrane is a single, host-derived membrane, whereas the remainder of the underlying apparent membranes surrounding the algal cell are symbiont-derived. The potential for macromolecules associated with these membranes to act as cellular signals critical to recruiting symbionts and maintaining established symbioses is discussed.

Larval development and metamorphosis in the nudibranch Melibe leonina (Gould) are described from observations of living animals and from one micrometer histological sections. Larval morphogenesis is similar to that previously described for other species of planktotrophic opisthobranch larvae except the rudiments of the primary cerata and the oral hood of the post-metamorphic stage appear in the late stage larva. Unlike many other opisthobranch larvae, M. leonina does not appear to require a specific exogenous cue to induce metamorphosis. Metamorphosis involves loss of the shell, operculum, velar ciliated cells, and certain components of the larval stomach but the left and right digestive diverticula are retained. A rapid expansion of the primary cerata and the oral hood occurs and is accompanied by a large volume increase of the internal hemocoel of these structures and a flattening and vesiculation of their epithelial cells. Several neuronal somata within the pleural ganglia become notably larger than their neighbors during metamorphosis. At approximately 2.5 days after shell loss, M. leonina begins to employ the oral hood to capture ciliates and small benthic nauplii. Morphogenesis in M. leonina is compared to that of other opisthobranchs and the premetamorphic appearance of the cerata and the lack of an exogenous metamorphic trigger are discussed.

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Adult Berghia verrucicornis individuals lay white, spiral egg masses containing zygotes. Egg masses are easily cultured in aerated, Millipore-filtered, seasoned aquarium water. Development proceeds quickly, with the bilobed velum apparent by the end of the second day, and the larval shell appearing at the beginning of the third day after oviposition. Hatching occurs 11 to 12 days after oviposition (23.9 +/- 1.3°C). If egg masses are incubated without aeration, poecilogonous development is observed; both larvae and juveniles hatch from the same undisturbed egg mass. The larvae metamorphose soon after hatching, losing the velum and larval shell. A habitat-specific inducer is not required for metamorphosis; but a factor associated with the sea anemone Aiptasia pallida appears to enhance a larva's tendency to metamorphose. Juveniles begin feeding on A. pallida three to four days after metamorphosis. Reproductive maturity is achieved as early as 47 days after oviposition. Because B. verrucicornis can be cultured, along with its prey A. pallida, at inland facilities, this nudibranch species may be a useful model for laboratory-oriented life history and neurobiological investigations.

Abstract:The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis. Keywords: Apoptosis -caenogastropod -mollusc -nitric oxide -serotonin Article: INTRODUCTION Larval Ilyanassa obsoleta, like most marine molluscs, undergo a series of physiological and anatomical transformations at metamorphosis which allow them to begin their juvenile life history phase. The most obvious external change is the loss of the ciliated larval feeding organ or velum [39], which, in the laboratory, occurs with a delay of some 12-36 hours after exposure to an inducing substance. In addition to loss of the velum, within 48 hours of metamorphic induction, internal transformations include rearrangements within the digestive tract and nervous system [13,14,27]. However, until recently, few changes in larval physiology or morphology during the 12-36 hour delay period in I. obsoleta had been described. In this species, by about 83% of larval development [27,42], the CNS contains rudiments of all of the adult ganglia along with a medial, unpaired apical ganglion (AG). Typically, the larval AG innervates the muscular and ciliary components of the velar lobes [20, 29-31, 34, 35]. The AG is an outgrowth of the trochophore apical tuft and has long been postulated to have a sensory function [6,34,35,38]. Recent experiments on a nudibranch provide evidence that the AG can detect a metamorphic cue [17], but a review of literature about molluscan AGs suggests that they are sensorimotor, coordinating the functions of the velar lobes [34,35] and sensing inductive and other stimuli [34]. Currently, the AG appears to be the only part of the larval CNS that is lost at metamorphosis. Some evidence from I. obsoleta and other species suggests t...

We reported a development of murine monoclonal antibodies to a molluscan small cardioactive peptide (SCPB) and their application to immunolabeling of neurons in several molluscan and arthropod species. In vitro stimulations of mouse lymphocytes with SCPB conjugated to a carrier protein yielded exclusively IgM class antibodies; in vivo stimulation resulted in generation of both IgM and IgG classes of antibodies. Monoclonal antibodies of the IgM class labeled identified SCP-containing neuron B11 in the frozen sections of the buccal ganglia of Tritonia diomedia. These antibodies failed to stain any neurons in whole mount preparations. A monoclonal antibody of IgG1 subclass selectively labeled neurons in both frozen sections and whole mount preparations of diverse invertebrate species. Thus, neurons B11, B12, and GE1 and several other neurons of the buccal and gastroesophageal ganglia of T. diomedia bound the antibody, and a similar pattern of immunolabeling was found in the closely related gastropod Tritonia festiva. We also observed SCPB-like immunoreactivity in the central neurons of other nudibranch and pulmonate molluscs and in examples of insect (Acheta domesticus and Tehrmobia domestica) and crustacean (Semibalanus cariosus) classes of the Arthropoda. Our results suggest a specific pattern of distribution of SCPB-like immunoreactivity in the gastropod nervous system and a broad occurrence of SCPB-like antigenicity in the diverse invertebrates.

This investigation examines tubulin labeling associated with the apical ganglion in a variety of planktotrophic and lecithotrophic opisthobranch larvae. Emphasis is on the ampullary neurons, in which ciliary bundles within the ampulla are strongly labeled. The larvae of all but one species have five ampullary neurons and their associated ciliary bundles. The anaspid Phyllaplysia taylori, a species with direct development and an encapsulated veliger stage, has only four ampullary neurons. The cilia-containing ampulla extends to the pretrochal surface via a long, narrow canal that opens to the external environment through a very small pore (0.1 microm diameter). Cilia within the canal were never observed to project beyond the opening of the apical pore. The ampullary canals extend toward and are grouped with the ciliary tuft cells and remain in this location as planktotrophic larvae feed and grow. If, as has been reported, the ciliary tuft is motile, the pores may be continually bathed in fresh seawater. Such an arrangement would increase sensitivity to environmental chemical stimuli if the suggested chemosensory function of these neurons is correct. In general, ciliary bundles of newly hatched veligers are smaller in planktotrophic larvae than in lecithotrophic larvae. In planktotrophic larvae of Melibe leonina, the ciliary bundles increase in length and width as the veligers feed and grow. This may be related to an increase in sensitivity for whatever sensory function these neurons fulfill. An unexpected tubulin-labeled structure, tentatively called the apical nerve, was also found to be associated with the apical ganglion. This putative nerve extends from the region of the visceral organs to a position either within or adjacent to the apical ganglion. One function of the apical nerve might be to convey the stimulus resulting from metamorphic induction to the visceral organs.