Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria

The anatomy and cellular organization of serotonergic neurons in the echinoderm apical organ exhibits class-specific features in dipleurula-type (auricularia, bipinnaria) and pluteus-type (ophiopluteus, echinopluteus) larvae. The apical organ forms in association with anterior ciliary structures. Apical organs in dipleurula-type larvae are more similar to each other than to those in either of the pluteus forms. In asteroid bipinnaria and holothuroid auricularia the apical organ spans ciliary band sectors that traverse the anterior-most end of the larvae. The asteroid apical organ also has prominent bilateral ganglia that connect with an apical network of neurites. The simple apical organ of the auricularia is similar to that in the hemichordate tornaria larva. Apical organs in pluteus forms differ markedly. The echinopluteus apical organ is a single structure on the oral hood between the larval arms comprised of two groups of cells joined by a commissure and its cell bodies do not reside in the ciliary band. Ophioplutei have a pair of lateral ganglia associated with the ciliary band of larval arms that may be the ophiuroid apical organ. Comparative anatomy of the serotonergic nervous systems in the dipleurula-type larvae of the Ambulacraria (Echinodermata+Hemichordata) suggests that the apical organ of this deuterostome clade originated as a simple bilaterally symmetric nerve plexus spanning ciliary band sectors at the anterior end of the larva. From this structure, the apical organ has been independently modified in association with the evolution of class-specific larval forms.     

A developmental study of serotonin-immunoreactive neurons in the larval central nervous system of the spider crabHyas araneus (Decapoda,Brachyura)

Larval development in crabs is characterized by a striking double metamorphosis in the course of which the animals change from a pelagic to a benthic life style. The larval central nervous system has to provide an adequate behavioural repertoire during this transition. Thus, processes of neuronal reorganization and refinement of the early larval nervous system could be expected to occur in the metamorphosing animal. In order to follow identified sets of neurons throughout metamorphosis, whole mount preparations of the brain and ventral nerve cord of laboratory reared spider crab larvae (Hyas araneus) were labelled with an antibody against the neurotransmitter serotonin. The system of serotonin-immunoreactive cell bodies, fibres and neuropils is well-developed in newly hatched larvae. Most immunoreative structures are located in the protocerebrum, with fewer in the suboesophaegeal ganglia, while the thoracic and abdominal ganglia initially comprise only a small number of serotonergic neurons and fibres. However, there are significant alterations in the staining pattern through larval development, some of which are correlated to metamorphic events. Accordingly, new serotonin-immunoreactive cells are added to the early larval set and the system of immunoreactive fibres is refined. These results are compared to the serotonergic innervation in other decapod crustaceans.     

Evolution of larval form in ophiuroids: insights from the metamorphic phenotype of Ophiothrix (Echinodermata: Ophiuroidea)

Comparison of development through metamorphosis in Ophiothrix species provided insights into the evolutionary relationships between Type I (ophiopluteus only) and Type II (ophiopluteus and vitellaria) patterns of development in the Ophiuroidea. As typical of Type I developers, the six inner larval arms in Ophiothrix spongicola were fully resorbed at metamorphosis and no remnants of ciliated epithelia were retained. The postero-lateral arms function as locomotory organs for the developing juvenile and were discarded at settlement. In contrast, in O. ciliaris the epithelia of the inner arms were transformed into ciliated ridges, similar to those seen in vitellariae and the postero-lateral arms were resorbed rather than being discarded. Larval arm resorption in O. ciliaris is similar to that in Type II developers. The metamorphic phenotype of O. ciliaris provides a link between Type I and II development. The presence of two types of metamorphosis in congeneric ophiuroids and the variable metamorphic phenotype of O. ciliaris was unexpected. It appears that closely related ophiuroids and individual species may have the capacity to metamorphose using either Type I or Type II pathways. Although the phylogenetic distribution of metamorphic phenotypes indicates that Type II development may be the ancestral state, comparative morphology suggests that a developmental dichotomy based on larval arm resorption may not be appropriate for the Ophiuroidea. Until metamorphosis is characterized for more taxa, the ancestral developmental mode for the Ophiuroidea will remain a matter of conjecture.     

Histochemical survey of transmitters in the central ganglia of the gastropod mollusc Phestilla sibogae

The aeolid nudibranch Phestilla sibogae is well studied in terms of its larval nervous system and neuronal involvement in metamorphosis. Central neurones in the adult have also been identified anatomically and electrophysiologically. We describe the neurotransmitter contents of these neurones and provide details of neuritic projections and developmental changes during growth (3 to 18 mm body length). Central ganglia from specimens of all sizes contained 100-115 serotonin-immunoreactive neurones, some of which appeared to be homologues of cells identified in other gastropods. Tyrosine hydroxylase immunoreactivity and aldehyde-induced fluorescence marked a common set of 28-30 catecholaminergic neurones located anteriorly in the cerebropleural ganglia and laterally in the pedal ganglia. Ganglionic neuropile and nerve trunks also contained many catecholaminergic fibres. About 65-100 intensely labelled FMRFamide-immunoreactive neurones were located symmetrically throughout the central ganglia, although one population was located only in the right pedal ganglion. Another 40-45 FMRFamide-immunoreactive neurones were weakly or variably stained. Central ganglia also contained 27-29 intensely labelled pedalpeptide-immunoreactive neurones, including those that were apparently homologues of cells previously described in Tritonia diomedea, and 16-19 weakly labelled pedal-peptide-immunoreactive neurones, including giant cerebropleural neurones coexhibiting FMRFamide immunoreactivity. Little cell addition involving any transmitter phenotype occurred as animals grew in body length, body growth being accommodated by growth in the size of individual cells, consistent with an approximate doubling in the size of the ganglia themselves.     

Serotonin immunoreactivity in the nervous system of the free‐swimming larvae and sessile adult females of Stephanoceros fimbriatus (Rotifera: Gnesiotrocha)

Unlike most rotifers (Rotifera), which are planktonic and direct developers, many gnesiotrochan rotifers (Monogononta: Gnesiotrocha) are sessile and have indirect development. Few details exist on larval metamorphosis in most gnesiotrochans, and considering the drastic transformation that takes place at metamorphosis—the replacement of the ciliated corona with a new head that bears ciliated tentacles (the infundibulum)—it is perhaps surprising that there are limited data on the process. Here, we document part of this metamorphosis by examining the presence and distribution of neurons with serotonin immunoreactivity in the nervous system of both planktonic larvae and sessile adult females. Using antibodies against serotonin combined with confocal laser‐scanning microscopy (CLSM) and 3D reconstruction software, we mapped the immunoreactive cell bodies and neurites in both life stages and found that relatively few changes occurred during metamorphosis. The larvae possessed a total of eight perikarya with serotonergic immunoreactivity (5HT‐IR) in the brain, with at least two pairs of perikarya outside the brain in the region of the corona. Cells with 5HT‐IR in the brain innervated the larval corona and also sent neurites to the trunk via the nerve cords. During metamorphosis, the corona was replaced by the infundibulum, which emerged from the larval mouth to become the new functional head. This change led to a posterior displacement of the brain and also involved the loss of 5HT‐IR in the lateral brain perikarya and the gain of two perikarya with 5HT‐IR in the anterior brain region. The innervation of the anterior end was retained in the adult; neurites that extended anteriorly to the mouth of the larva formed a distinct neural ring that encircled the infundibulum after metamorphosis. Significantly, there was no innervation of the infundibular tentacles by neurites with 5HT‐IR, which suggests that ciliary control is unlikely to be modulated by serotonin within the tentacles themselves.     

The development of the serotonergic and FMRF-amidergic nervous system in<Emphasis Type="Italic"> Antalis entalis</Emphasis> (Mollusca,Scaphopoda)

The morphogenesis of serotonin- and FMRF-amide-bearing neuronal elements in the scaphopod Antalis entalis was investigated by means of antibody staining and confocal laser scanning microscopy. Nervous system development starts with the establishment of two initial, flask-like, serotonergic central cells of the larval apical organ. Slightly later, the apical organ contains four serotonergic central cells which are interconnected with two lateral serotonergic cells via lateral nerve projections. At the same time the anlage of the adult FMRF-amide-positive cerebral nervous system starts at the base of the apical organ. Subsequently, the entire neuronal complex migrates behind the prototroch and the six larval serotonergic cells lose transmitter expression prior to metamorphic competence. There are no strictly larval FMRF-amide-positive neuronal structures. The development of major adult FMRF-amide-containing components such as the cerebral system, the visceral loop, and the buccal nerve cords, however, starts before the onset of metamorphosis. The anlage of the putative cerebral system is the only site of adult serotonin expression in Antalis larvae. Establishment of the adult FMRF-amidergic and serotonergic neuronal bauplan proceeds rapidly after metamorphosis. Neurogenesis reflects the general observation that the larval phase and the expression of distinct larval morphological features are less pronounced in Scaphopoda than in Gastropoda or Bivalvia. The degeneration of the entire larval apical organ before metamorphic competence argues against an involvement of this sensory system in scaphopod metamorphosis. The lack of data on the neurogenesis in the aplacophoran taxa prevent a final conclusion regarding the plesiomorphic condition in the Mollusca. Nevertheless, the results presented herein shed doubts on general theories regarding possible functions of larval "apical organs" of Lophotrochozoa or even Metazoa.     

Development of the nervous system in the brittle star Amphipholis kochii

There are several studies of neural development in various echinoderms, but few on ophiuroids, which develop indirectly via the production of pluteus larvae, as do echinoids. To determine the extent of similarity of neuroanatomy and neural development in the ophiuroids with other echinoderm larvae, we investigated the development of the nervous system in the brittle star Amphipholis kochii (Echinodermata: Ophiuroidea) by immunohistochemistry. Immunoreactive cells first appeared bilaterally in the animal pole at the late gastrula stage, and there was little migration of the neural precursors during A. kochii ontogeny, as is also the case in echinoids and holothuroids. On the other hand, neural specification in the presumptive ciliary band near the base of the arms does occur in ophiuroid larvae and is a feature they share with echinoids and ophiuroids. The ophiopluteus larval nervous system is similar to that of auricularia larvae on the whole, including the lack of a fine network of neurites in the epidermis and the presence of neural connections across the oral epidermis. Ophioplutei possess a pair of bilateral apical organs that differ from those of echinoid echinoplutei in terms of relative position. They also possess coiled cilia, which may possess a sensory function, but in the same location as the serotonergic apical ganglia. These coiled cilia are thought to be a derived structure in pluteus-like larvae. Our results suggest that the neural specification in the animal plate in ophiuroids, holothuroids, and echinoids is a plesiomorphic feature of the Ambulacraria, whereas neural specification at the base of the larval arms may be a more derived state restricted to pluteus-like larvae.     

Structure and metamorphic remodeling of the larval nervous system and musculature of Phoronis pallida (Phoronida)

The structure of the larval nervous system and the musculature of Phoronis pallida were studied, as well as the remodeling of these systems at metamorphosis. The serotonergic portion of the apical ganglion is a U-shaped field of cell bodies that send projections into a central neuropil. The majority of the serotonergic cells are (at least) bipolar sensory cells, and a few are nonsensory cells. Catecholaminergic cell bodies border the apical ganglion. The second (hood) sense organ develops at competence and is composed of bipolar sensory cells that send projections into a secondary neuropil. Musculature of the competent larva includes circular and longitudinal muscle fibers of the body wall, as well as elevators and depressors of the tentacles and hood. The juvenile nervous system and musculature are developed prior to metamorphosis and are integrated with those of the larva. Components of the juvenile nervous system include a diffuse neural net of serotonergic cell bodies and fibers and longitudinal catecholaminergic fibers. The juvenile body wall musculature consists of longitudinal fibers that overlie circular muscle fibers, except in the cincture regions, where this pattern is reversed. Metamorphosis is initiated by the larval neuromuscular system but is completed by the juvenile neuromuscular system. During metamorphosis, the larval nervous system and the musculature undergo cell death, and the larval tentacles and gut are remodeled into the juvenile arrangement. Although the phoronid nervous system has often been described as deuterostome-like, these data show that several cytological aspects of the larval and juvenile neuromuscular systems also have protostome (lophotrochozoan) characteristics.     

Development of serotonin-like and SALMFamide-like immunoreactivity in the nervous system of the sea urchin Psammechinus miliaris.

The present immunocytochemical study utilizes serotonin and SALMFamide antisera, together with confocal laser scanning microscopy, to provide new information about the development of the nervous system in the sea urchin Psammechinus miliaris (Echinodermata: Echinoidea). Special attention is paid to the extent of the nervous system in later larval stages (6-armed pluteus to metamorphic competency), a characteristic that has not been well described in this and other species of sea urchin. An extensive apical ganglion appears by the 6-armed pluteus stage, forming a complex of 10-20 cells and fibers, including discrete populations of both serotonin-like and SALMF-amide-like immunoreactive cells. At metamorphosis this complex is large, comprising at least 40 cells in distinct arrays. Serotonin-like immunoreactivity is also particularly apparent in the lower lip ganglion of 6- to 8-armed plutei; this ganglion consists of 15-18 cells that are distributed around the mouth. The ciliary nerves that lie beneath the ciliary bands in the larval arms, the esophagus, and a hitherto undescribed network associated with the pylorus all show SALMFamide-like immunoreactivity. The network of cells and fibers in the pyloric area develops later in larval life. It first appears as one cell body and fiber, then increases in size and complexity through the 8-armed pluteus stage to form a complex of cells that encircles the pylorus. SALMFamide-like, but not serotonin-like, immunoreactivity is seen in the vestibule wall, tube feet, and developing radial nerve fibers of the sea urchin adult rudiment as the larva gains metamorphic competency.     

Larval and adult brains

Apical organs are a well-known structure in almost all ciliated eumetazoan larvae, although their function is poorly known. A review of the literature indicates that this small ganglion is the "brain" of the early larva, and it seems probable that it represents the brain of the ancestral, holopelagic ancestor of all eumetazoans, the gastraea. This early brain is lost before or at metamorphosis in all groups. Protostomes (excluding phoronids and brachiopods) appear to have brains of dual origin. Their larvae develop a pair of cephalic ganglia at the episphere lateral to the apical organ, and these two ganglia become an important part of the adult brain. The episphere and the cerebral ganglia show Otx expression, whereas Hox gene expression has not been seen in this part of the brain. A ventral nervous system develops around the blastopore, which becomes divided into mouth and anus by fusion of the lateral blastopore lips. The circumblastoporal nerve ring becomes differentiated into a nerve ring around the mouth, becoming part of the adult brain, a pair of ventral nerve cords, in some cases differentiated into a chain of ganglia, and a ring around the anus. This part of the nervous system appears to be homologous with the oral nerve ring of cnidarians. This interpretation is supported by the expression of Hox genes around the cnidarian mouth and in the ventral nervous system of the protostomes. The development of phoronids, brachiopods, echinoderms, and enteropneusts does not lead to the formation of an episphere or to differentiation of cerebral ganglia. In general, a well-defined brain is lacking, and Hox genes are generally not expressed in the larval organs, although this has not been well studied.     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

The structure of the larval nervous system of Pisaster ochraceus (Echinodermata: Asteroidea)

Ultrastructural observations and glyoxilic acid-induced fluorescence of catecholamines indicate that tracts of axons lie at the base of the ciliary bands and run throughout their length in bipinnaria and brachiolaria larvae of Pisaster ochraceus. Two types of nerve cells occur at regular intervals within the ciliary bands. Type I nerve cells are associated with the axonal tracts, and type II nerve cells, which are ciliated, occur along the edge of the ciliary bands. Two prominent ganglia, which appear as accumulations of nerve cells and neuropile, occur on the lower lip of the larval mouth. Smaller ganglia occur irregularly throughout the ciliary band. Synapses were never clearly identified and were assumed to be unspecialized. Nervous tissues were also found associated with the esophageal muscles, the attachment organ, and the larval arms. Organization of the nervous system and its association with effectors suggest it controls swimming and feeding. Several similarities exist between the nervous systems of larval asteroids, larval echinoids, and adult echinoderms.     

Constancy and variation of the serotonin-like immunoreactive neurons in the metamorphosing ventral nerve cord of the meal beetle,Tenebrio molitor L. (Coleoptera : Tenebrionidae)

Serotonin-like immunoreactive neurons were mapped in the larval, prepupal, pupal, and adult ventral nerve cord (VNC) of the beetle, Tenebrio molitor L. (Coleoptera: Tenebrionidae). The alterations of the shape of these neurons during metamorphosis were analysed. The stage-specific interindividual variability of the examined serotonin-like immunoreactive neurons is low. Serotonin-like immunoreactive neurons of the abdominal and thoracic ganglia behave differently during metamorphosis. Only in thoracic ganglia was an obvious change in the pattern of serotonin-like immunoreactive neurons observed. The shape of the dendritic trees of serotonin-like immunoreactive neurons varies in thoracic., but not in abdominal ganglia. During postlarval development, new emerging neurons that react with the anti-serotonin antibody are found only in the thoracic ganglia. Serotonin-like immunoreactive neurons are serially homologous in the larval ventral nerve cord. The basic organization of the serotonin-like immunoreactive neurons is maintained up to the adult stage. Some aspects of the metamorphosis of the nervous system are discussed with respect to the transformation of the set of immunoreactive neurons from larval to adult stage. The results are compared to those obtained in the study of serotonin-immunoreactive neurons in cockroaches, dipterans and locusts.     

Evolutionary Conservation of the Larval Serotonergic Nervous System in a Direct Developing Sea Urchin

Development of the larval serotonergic nervous system is examined by indirect immunofluorescence in two congeneric species of sea urchins that exhibit divergent embryonic and larval development. Heliocidar is tuberculata undergoes indirect planktotrophic development via a pluteus larva, whereas Heliocidaris erythrogramma develops directly, passing through a brief, highly derived lecithotrophic larval stage. We have cleared the opaque embryos of H. erythrogramma and discuss internal features of its development. The serotonergic nervous system of H. tuberculata arises in the apical plate at the end of gastrulation and develops into a bilaterally symmetric ganglion lying between the anterolateral arms in the preoral hood. Putatively homologous neurons appear at the apical end of the modified larva of H. erythrogramma well after the completion of gastrulation, coincident with development of the primary podia of the adult rudiment. The neurons form a bilaterally symmetric ganglion whose orientation relative to the vestibule is conserved with respect to that found in planktotrophic larvae. This allows us to define a left and right side for this larva which lacks external points of asymmetry such as a larval mouth. The alteration in the time of nervous system development in H. erythrogramma relative to that of H. tuberculata , and other indirect developers, implicates heterochronies in cellular differentiation as an important component of the evolution of direct development.     

GABA is an inhibitory neurotransmitter in the neural circuit regulating metamorphosis in a marine snail

The marine mud snail, Tritia (=Ilyanassa) obsoleta, displays a biphasic life cycle. During the initial phase of early development, embryos hatch from benthic egg capsules to become weakly swimming veliger larvae. In the second phase, adult T. obsoleta are facultative carnivores and major agents of community disturbance. Metamorphosis is the irreversible developmental event that links these two life history stages. When physiologically competent, larvae can respond to appropriate environmental cues by settling onto their mudflat habitat and transforming themselves into miniature adult snails. Two neurotransmitters—serotonin and nitric oxide—have opposing effects on the metamorphic process in this species. In multiple other species of gastropod and bivalve molluscs, a third neurotransmitter, the classically inhibitory compound γ‐aminobutyric acid (GABA), can induce settlement or metamorphosis upon external application to competent larvae. In this situation, GABA is presumed to mimic the action of ligands from the juvenile environment that bind to larval chemosensory receptors and activate the metamorphic pathway. Results of our experiments contradict this commonly reported action of GABA on molluscan larvae. External application of GABA to competent larvae of T. obsoleta elicited no response, but instead attenuated the action of serotonin (5‐HT), a metamorphic inducer. Our investigations into the responses of larval T. obsoleta to multiple GABAergic reagents support our hypothesis that GABA functions internally as a neurotransmitter in the pathway that controls the initiation of metamorphosis. Our results also suggest that GABA acts directly on or downstream from serotonergic neurons to regulate the metamorphosis‐inducing effects of this neurotransmitter. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 736–753, 2018     

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Summary Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.