Behavior,metamorphosis, and muscular organization of the predatory rotifer Acyclus inquietus (Rotifera,Monogononta)

Abstract. The atrochid rotifer, Acyclus inquietus, is a sedentary predator that lives within the colonies of its prey, the rotifer Sinantherina socialis. After larvae infiltrate and become associated with the colony, they secrete a permanent gelatinous tube and undergo metamorphosis to the adult stage. We followed settlement and metamorphosis using bright-field microscopy to document specific larval behaviors after eclosion, and used epifluorescence and confocal microscopy of phalloidin-labeled specimens to visualize some of the morphological changes that occur during metamorphosis. Upon eclosion, larvae possess paired eyespots and a ciliated corona that functions strictly in locomotion. After leaving the parent's gelatinous tube, larvae eventually settle on unoccupied colonies of S. socialis or on other substrates if colonies are unavailable. Settlement involves a period of gliding among colony members before attachment with the foot and the secretion of a gelatinous tube. After settlement, there is a drastic reconfiguration of the corona that involves loss of the eyespots, loss of the coronal cilia, and the formation of the cup-shaped infundibulum, a deep depression in the anterior of the head that leads to the mouth. The development of the infundibulum involves the expansion of tissues around the mouth and is accompanied by a reorientation of the underlying musculature that supplies the infundibulum and allows its use in prey capture. The arrangement of the muscles in the trunk and foot regions, which contain outer circular (complete and incomplete) and inner longitudinal bands, remains unchanged between ontogenetic stages, and reflects the condition characteristic of other rotifers.     

Inter‐ and intraspecific plasticity in distribution patterns of immunoreactive compounds in actinotroch larvae of Phoronida (Lophotrochozoa)

Recent molecular data place Phoronida within the protostome superclade Lophotrochozoa, where they have been suggested to form a monophyletic assemblage with Brachiopoda and/or Nemertea. Herein, the anatomy of the nervous system and the structure of the apical organ are described for two larval stages of Phoronis muelleri in order to contribute to the discussion concerning the evolution of lophotrochozoan nervous systems. Specimens were investigated by confocal laser scanning microscopy using antibodies against the serotonin‐like immunoreactive (serotonin‐lir), the FMRF‐amide‐like immunoreactive (FMRFamide‐lir) and the small cardioactive peptide B‐like immunoreactive (small cardioactive peptide B‐lir) compounds of the nervous system. Consistent with larvae of other phoronid species, we found a complex apical organ that consists of numerous serotonin‐lir flask‐shaped cells, additional bi‐ or multipolar serotonin‐lir cells and several FMRFamide‐lir perikarya. A detailed comparison between our results and those of a previous study on the same species shows significant differences in the innervation of the preoral lobe, the tentacles and the telotroch. Our work is the first to prove the presence of small cardioactive peptide B in phoronids. In larvae of P. muelleri, it is expressed in neurites along the margin of the preoral hood, in the mesosome, in the tentacles, in the trunk as well as in the apical organ. A positive signal for this peptide is also known from molluscs, annelids and arthropods, indicating that it was also part of the protostomian groundplan. In contrast to a recent study on another phoronid species, Phoronopsis harmeri, we did not find a ventral neurite bundle in the larval stages investigated herein, thus leaving the question open whether this structure was part of the phoronid groundplan or evolved de novo in P. harmeri.     

Three‐dimensional reconstruction and neural map of the serotonergic brain of Asplanchna brightwellii (Rotifera,Monogononta)

The basic organization of the rotifer brain has been known for nearly a century; yet, fine details on its structure and organization remain limited despite the importance of rotifers in studies of evolution and population biology. To gain insight into the structure of the rotifer brain, and provide a foundation for future neurophysiologic and neurophylogenetic research, the brain of Asplanchna brightwellii was studied with immunohistochemistry, confocal laser scanning microscopy, and computer modeling. A three‐dimensional map of serotonergic connections reveals a complex network of approximately 28 mostly unipolar, cerebral perikarya and associated neurites. Cells and their projections display symmetry in quantity, size, connections, and pathways between cerebral hemispheres within and among individuals. Most immunopositive cells are distributed close to the brain midline. Three pairs of neurites form decussations at the brain midline and may innervate sensory receptors in the corona. A single neuronal pathway appears to connect both the lateral horns and dorsolateral apical receptors, suggesting that convergence of synaptic connections may be common in the afferent sensory systems of rotifers. Results show that the neural map of A. brightwellii is much more intricate than that of other monogonont rotifers; nevertheless, the consistency in neural circuits provides opportunities to identify homologous neurons, distinguish functional regions based on neurotransmitter phenotype, and explore new avenues of neurophylogeny in Rotifera. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

On the serotonergic nervous system of two planktonic rotifers, Conochilus coenobasis and C. dossuarius (Monogononta, Flosculariacea, Conochilidae)

The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the first immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epifluorescence and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and C. dossuarius, the serotonergic nervous system is defined by a dorsal cerebral ganglion, apically directed cerebral neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT-immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical field. The most dorsal pair innervates a coronal nerve ring that encircles the apical field. Within the apical field is a second nerve ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Specific differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta.     

Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

Background

Inferences concerning the evolution of invertebrate nervous systems are often hampered by the lack of a solid data base for little known but phylogenetically crucial taxa. In order to contribute to the discussion concerning the ancestral neural pattern of the Lophotrochozoa (a major clade that includes a number of phyla that exhibit a ciliated larva in their life cycle), we investigated neurogenesis in Phoronopsis harmeri, a member of the poorly studied Phoronida, by using antibody staining against serotonin and FMRFamide in combination with confocal microscopy and 3D reconstruction software.

Results

The larva of Phoronopsis harmeri exhibits a highly complex nervous system, including an apical organ that consists of four different neural cell types, such as numerous serotonin-like immunoreactive flask-shaped cells. In addition, serotonin- and FMRFamide-like immunoreactive bi- or multipolar perikarya that give rise to a tentacular neurite bundle which innervates the postoral ciliated band are found. The preoral ciliated band is innervated by marginal serotonin-like as well as FMRFamide-like immunoreactive neurite bundles. The telotroch is innervated by two neurite bundles. The oral field is the most densely innervated area and contains ventral and ventro-lateral neurite bundles as well as several groups of perikarya. The digestive system is innervated by both serotonin- and FMRFamide-like immunoreactive neurites and perikarya. Importantly, older larvae of P. harmeri show a paired ventral neurite bundle with serial commissures and perikarya.

Conclusions

Serotonin-like flask-shaped cells such as the ones described herein for Phoronopsis harmeri are found in the majority of lophotrochozoan larvae and therefore most likely belong to the ground pattern of the last common lophotrochozoan ancestor. The finding of a transitory paired ventral neurite bundle with serially repeated commissures that disappears during metamorphosis suggests that such a structure was part of the ??ur-phoronid?? nervous system, but was lost in the adult stage, probably due to its acquired sessile benthic lifestyle.     

Peptidergic and serotonergic immunoreactivity in the metamorphosing ophiopluteus of Ophiactis resiliens (Echinodermata, Ophiuroidea)

Abstract. Antibodies against the echinoderm-specific neuropeptide S1 and against 5HT were used to examine the fate of the larval nervous system during metamorphosis in the ophiuroid Ophiactis resiliens . In contrast to most echinoderms, the onset of peptidergic and serotonergic expression was delayed to the advanced ophiopluteus stage, in particular for 5HT. In advanced ophioplutei, peptidergic immunoreactivity was located in simple fibres associated with the ciliated bands, a stomach nerve ring, and cells along the antero-lateral arms. 5HT immunoreactivity was concentrated in 2 oral ganglia in the adoral projections, located at the posterior rim of the mouth. Clusters of 5HT-positive cells were also found along the antero-lateral arms. The ophiopluteus lacked a serotonergic (or peptidergic) anterior ganglion. In echinoids, holothuroids, and crinoids, anterior ganglia are thought to have a sensory role in settlement and metamorphosis. Given that ophioplutei metamorphose in the plankton and that larval structures degenerate before settlement, the absence of apical ganglia correlates with the lack of a functional role for larval structures in substrate selection and settlement. Although most of the larval nervous system degenerated during metamorphosis, the adoral projections and associated oral ganglia appeared to be incorporated into the juvenile mouth, suggesting a potential role for larval neurons in contributing to oral neuronal structures in the adult. S1-positive neurons and fibres in the rudiment developed de novo and in parallel with development of the epineural canal. This structure gives rise to the primordia of the adult circumoral nerve ring and radial nerves, indicating that differentiation of the adult nervous system begins in the early stages of metamorphosis.     

Serotonin and Nitric Oxide Regulate Metamorphosis in the Marine Snail Ilyanassa obsoleta

Several neuroactive compounds have been implicated as playingroles in the circuitry that controls larval metamorphosis inmarine molluscs. For the caenogastropod Ilyanassa obsoleta,results of neuroanatomical studies suggest that the productionof nitric oxide (NO) increases throughout the planktonic stageand that NO production is necessary for the maintenance of thelarval state, especially as it becomes metamorphically competent.Bath application or injection of exogenous serotonin (5HT) caninitiate metamorphosis in competent larvae, and exogenous NOcan inhibit such serotonergically-induced metamorphosis. Inhibitionof endogenous nitric oxide synthase (NOS) can also trigger larvalmetamorphosis. The production of endogenous NO appears to decreaseconcurrently with the initiation of metamorphosis, but the specificinteractions between serotonergic and nitrergic neurons areunknown. Evidence in support of NO acting to up-regulate theenzyme guanylyl cyclase (GC) is still equivocal. Thus, we donot yet know if NO exerts its effects through the actions ofcyclic 3',5'-guanosine monophosphate (cGMP) or by a cGMP-independentmechanism. The ubiquity of nitrergic signalling and its significancefor developing molluscan embryos and larvae are still the subjectof speculation and require further investigation.     

Programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc Ilyanassa obsoleta

The apical ganglion (AG) of larval caenogastropods, such as Ilyanassa obsoleta, houses a sensory organ, contains five serotonergic neurons, innervates the muscular and ciliary components of the velum, and sends neurites into a neuropil that lies atop the cerebral commissure. During metamorphosis, the AG is lost. This loss had been postulated to occur through some form of programmed cell death (PCD), but it is possible for cells within the AG to be respecified or to migrate into adjacent ganglia. Evidence from histological sections is supported by results from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicate that cells of the AG degenerate by PCD. PCD occurs after metamorphic induction by serotonin or by inhibition of nitric oxide synthase (NOS) activity. Cellular degeneration and nuclear condensation and loss were observed within 12 h of metamorphic induction by NOS inhibition and occur before loss of the velar lobes, the ciliated tissue used for larval swimming and feeding. Velar disintegration happens more rapidly after metamorphic induction by serotonin than by 7-nitroindazole, a NOS inhibitor. Loss of the AG was complete by 72 h after induction. Spontaneous loss of the AG in older competent larvae may arise from a natural decrease in endogenous NOS activity, giving rise to the tendency of aging larvae to display spontaneous metamorphosis in culture.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Serotonin immunoreactivity in the nervous system of the Pandora larva, the Prometheus larva, and the dwarf male of Symbion americanus (Cycliophora)

Cycliophora is a recently described phylum of enigmatic metazoans with a very complex life cycle that includes several sexual and asexual stages. Symbion pandora and Symbion americanus are the only two cycliophoran species hitherto described, of which morphological and genetic knowledge is still deficient to clarify the phylogenetic position of the phylum. Aiming to increase the database on the cycliophoran neural architecture, we investigated serotonin immunoreactivity in the free swimming Pandora larva, the Prometheus larva, and the adult dwarf male of S. americanus. In the larval forms, serotonin is mainly expressed in a ring-shaped pattern at the periphery of the antero-dorsal cerebral ganglion. Additionally, several serotonergic perikarya emerge from both sides of the cerebral ganglion. Thin neurites project anteriorly from the cerebral ganglion, while a pair of ventral longitudinal neurites emerges laterally and runs along the anterior-posterior body axis. Posteriorly, the ventral neurites fuse and extend as a posterior projection. In the dwarf male, serotonin is found mainly in the commissural neuropil of the large anterior cerebral ganglion. In addition, serotonin immunoreactivity is present in the most anterior region of the ventral neurites. Comparative analysis of spiralian nervous systems demonstrates that the neuroanatomy of the cycliophoran larval stages resembles much more the situation of adult rather than larval spiralians, which may be explained by secondary loss of larval structures and heterochronic shift of adult components into the nervous system of the Pandora and the Prometheus larva, respectively.     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

Observations of serotonin and FMRFamide-like immunoreactivity in palp sensory structures and the anterior nervous system of spionid polychaetes

Evidence suggests that ciliated sensory structures on the feeding palps of spionid polychaetes may function as chemoreceptors to modulate deposit-feeding activity. To investigate the probable sensory nature of these ciliated cells, we used immunohistochemistry, epi-fluorescence, and confocal laser scanning microscopy to label and image sensory cells, nerves, and their organization relative to the anterior central nervous system in several spionid polychaete species. Antibodies directed against acetylated alphatubulin were used to label the nervous system and detail the innervation of palp sensory cells in all species. In addition, the distribution of serotonin (5-HT) and FMRFamide-like immunoreactivity was compared in the spionid polychaetes Dipolydora quadrilobata and Pygospio elegans. The distribution of serotonin immunoreactivity was also examined in the palps of Polydora cornuta and Streblospio benedicti. Serotonin immunoreactivity was concentrated in cells underlying the food groove of the palps, in the palp nerves, and in the cerebral ganglion. FMRFamide-like immunoreactivity was associated with the cerebral ganglia, nuchal organs and palp nerves, and also with the perikarya of ciliated sensory cells on the palps.     

Neuromuscular organization of the freshwater colonial rotifer, Sinantherina socialis, and its implications for understanding the evolution of coloniality in Rotifera

Coloniality among rotifers is rare, and while the adaptive significance of the lifestyle has been explored previously, there are few details about how it may have influenced the morphology of colony members. In this study, we use confocal laser scanning microscopy combined with cyto- and immunohistochemistry to determine if the colonial rotifer, Sinantherina socialis, differs in neuromuscular organization relative to other colonial and solitary forms. Our observations indicate that the patterns of serotonergic neurons and somatic muscles in S. socialis are broadly similar to other rotifers. At the neuronal level, the distribution of serotonergic (5HT-IR) neurons in S. socialis is most similar to colony-forming species of Conochilus: (1) paired nerve cords extend the length of the body; (2) the cerebral ganglion innervates the corona with paired neurites that form a neuronal ring; and (3) a single pair of neurites innervates the ventral sensory antennae. Unique to S. socialis is the presence of two additional neuronal rings that supply the corona and may function to modulate ciliary beat. At the muscular level, S. socialis displays a typical pattern of somatic muscle organization similar to other rotifers, but diverges from the pattern in three significant ways: (1) somatic circular muscles form complete rings; (2) circular muscles are distinguishable based on their size and position in the body; and (3) transverse muscles are present within the corona and presumably function to modify its shape for feeding and defensive purposes. These differences in neuromuscular organization may be adaptations to the colonial and/or sessile lifestyle, both of which are characteristic of S. socialis.     

First description of the serotonergic nervous system in a bdelloid rotifer: Macrotrachela quadricornifera Milne 1886 (Philodinidae)

Class Bdelloidea of phylum Rotifera comprises aquatic microinvertebrates that are known for both obligate parthenogenesis and for resisting desiccation through a dormant reversible state. In the frame of an investigation about the role of the nervous system in controlling life cycle, reproduction and dormancy, we describe the serotonergic system of a bdelloid, Macrotrachela quadricornifera, using serotonin immunohistochemistry and confocal laser scanning microscopy. Serotonin immunoreactivity is present in the cerebral ganglion, lateral nerve cords and peripheral neurites. The cerebral ganglion consists of perikarya that send neurites cephalically to the rostrum and corona. A pair of neurites exits the cerebral ganglion as lateral nerve cords, and proceeds caudally to the pedal ganglion where additional neurites enter the foot. Based on the location of serotonergic immunoreactivity, we hypothesize that the neurotransmitter is involved in both motor activity (e.g., ciliary beating, inchworm-like locomotion) and sensory activity. A comparison between the serotonergic nervous systems of M. quadricornifera and species of Monogononta reveals differences in the numbers and patterns of cerebral perikarya, peripheral perikarya, and periperhal neurites. These differences may have functional significance for understanding adaptations to specific environments and/or systematic significance for reconstructing the rotiferan ground pattern.     

Intraspinal serotonergic signaling suppresses locomotor activity in larval zebrafish

Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post‐fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally‐extending neurites resembling dendrites and a caudally‐extending neurite resembling an axon, which terminated with an enlarged growth cone‐like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally‐produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin‐2 each produced similar effects to 5HT, indicating that spinally‐intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

Effect of reserpine on 5-hydroxytryptophan (5HTP)-immunoreactive neurons in the rat brain

By immunohistochemistry of rat brain in conjunction with a specific antibody against 5-hydroxytryptophan (5HTP), we examined immunoreactivity to 5HTP in neurons, from which 5-hydroxytryptamine (5HT; serotonin) was depleted by reserpine treatment. The distribution patterns of 5HTP-positive neurons overlapped with those of 5HT neurons. Treatment with reserpine (5 mg/kg, 90 min before death) caused a complete suppression of 5HT-positive staining, but 5HTP-immunostaining remained in perikarya of the nuclei raphe dorsalis, centralis superior and obscurus. Treatment with reserpine (25 mg/kg, 90 min before death) suppressed the 5HTP-immunoreaction in certain perikarya (e.g. of the nucleus raphe dorsalis) and fibres; however, 5HTP-immunostaining remained in perikarya of the nuclei centralis superior and raphe obscurus. This suggests that these neurons synthesize more 5HTP by a process which appears to be stimulated by reserpine.     

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.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Waterborne compounds from the green seaweed Ulva reticulata as inhibitive cues for larval attachment and metamorphosis in the Polychaete Hydroides elegans

Field observations in Hong Kong waters have shown the marine macroalga Ulva reticulata Forsskal (Chlorophyta) to be free of fouling. In this study, the presumptive antifouling mechanism in this alga was investigated and attempts were made to distinguish between possible mechanisms. Waterborne algal compounds were analyzed in larval behavioral bioassays with the marine polychaete Hydroides elegans, a major fouling organism in tropical waters around the world. Larval attachment and metamorphosis in this sessile species is induced by specific bacterial strains in natural bio‐organic films. In bioassays with alga‐conditioned seawater, levels of larval metamorphosis were significantly reduced in comparison to controls. The results demonstrate that the inhibitive effect was caused by direct larval deterrence. Following a bio‐assay‐guided isolation procedure, an inhibitive fraction for larval metamorphosis was purified from U. reticulata‐conditioned seawater. Preliminary chemical analysis of the biologically active fraction pointed to polysaccharides, proteins or glycoconjugates with a molecular weight > 100 kD.     

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.