Serotonin Plays an Early Role in the Metamorphosis of the HydrozoanPhialidium gregarium

Hydrozoan larvae normally metamorphose in response to an obligate external environmental cue. Application of certain artificial chemical stimuli will also induce metamorphosis. These chemicals and their inhibitors have been used to define and order some of the signal transduction events involved in this process. Results from this study show that exogenous application of serotonin (5-HT) will induce metamorphosis and that 5-HT immunoreactive cells are present in larvae when they are competent to metamorphose. The 5-HT inhibitors ketanserin, clozapine, and 5,7-DHT prevent metamorphosis from occurring as a response to a natural inducing stimulus. Additionally, 5-HT signaling occurs prior to both an influx of external Ca²⁺from seawater and activation of protein kinase C, two other steps in the metamorphic signal transduction pathway. The neuropeptide LWamide, previously shown to induce metamorphosis in a related hydrozoan,Hydractinia echinata,also induced metamorphosis inPhialidium.When larvae were cotreated with LWamide and the 5-HT antagonist ketanserin, settlement occurred but was not followed by polyp morphogenesis. These results are used to present a model for the action of 5-HT during metamorphosis inPhialidium gregarium.     

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.     

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

Background

A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus.

Results

Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae.

Conclusions

We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom - metamorphosis.     

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.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10^?4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC₅₀) for chlorpromazine and amitriptyline was 1.6 × 10^?6 M and 6.6 × 10^?5 M, respectively. Idazoxan was less effective with an IC₅₀ of 4.4 × 10¹³ M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K⁺ was not inhibited by 10^?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

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.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

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.     

Development in Berthella californica (Gastropoda: Opisthobranchia) with comparative observations on phylogenetically relevant larval characters among nudipleuran opisthobranchs

Abstract. Morphological criteria defining the Nudipleura and sister group relationships among the three nudipleuran subgroups (pleurobranchoideans, anthobranch nudibranchs, and cladobranch nudibranchs) have been controversial. Analysis of larval stages may help resolve these uncertainties by identifying additional phylogenetically informative characters, but existing information on pleurobranchoidean larvae is meager. We studied larval development and metamorphosis of the pleurobranchoidean Berthella californica using histological sections, scanning and transmission electron microscopy, and immunolabeling of neurons within the larval apical ganglion. We also provide comparative data on other nudipleuran larvae that may be useful for phylogenetic reconstruction. Berthella californica fills a previously unoccupied place within an evolutionary scenario that derives nudibranchs from pleurobranchoideans, two groups in which the larval mantle fold forms the post‐metamorphic notum (dorsal epidermis). In B. californica, reflection of the mantle fold epithelium to form the notum begins at metamorphosis, as also occurs in nudibranchs, whereas mantle reflection in other pleurobranchoideans begins well before metamorphosis. Dissolution of overgrown shell walls inside the protoconch and formation of the post‐metamorphic notum from the inner epithelium of the larval mantle fold may be synapomorphies of the Nudipleura. The larval shell in B. californica is additionally noteworthy because it acquires bilateral symmetry later in development, which is very unusual among larval opisthobranchs. We demonstrate an osphradium in the larvae of two pleurobranchoideans and one anthobranch nudibranch, although adults lack this trait. We also identified an autapomorphy of cladobranch nudibranchs in the form of five ampullary neurons within the larval apical ganglion, whereas other planktotrophic opisthobranch larvae have only four of these neurons. Although our data provide morphological criteria defining both the Nudipleura and the cladobranch nudibranchs, they are insufficient to resolve sister group relationships within the Nudipleura.     

Skeletal morphogenesis in the urodele skull: II. Effect of developmental stage in thyroid hormone-induced remodeling

This study investigates the effect of developmental stage on thyroid hormone (TH)-mediated remodeling in the skeletal tissues of hemidactyliine plethodontid urodeles. Rate of morphogenesis was quantified in 17 metamorphic tissues for three different size-age classes of Eurycea bislineata larvae immersed in a metamorphic dosage of T₄. Extent of morphogenesis after a 3-week immersion was also quantified in these tissues plus four larval ones for the full size range of E. bislineata larvae and for less complete size ranges of E. wilderae, E. longicauda guttolineata, Gyrinophilus porphyriticus, and Pseudotriton ruber larvae. Although all tissues respond more slowly with decreasing size/age, two tissue-specific effects are evident in all species. Larval ossifications are less inducible than metamorphic ossifications, and progressive metamorphic events are more retarded and, in some cases, more prone to abnormal morphogenesis than regressive ones. The first effect agrees with the prediction that tissues that naturally remodel at metamorphosis are more responsive to a metamorphic dosage of TH than those that respond at a larval stage and lower TH. The second effect agrees with the prediction that progressive morphogenesis is more likely to be impaired at small size than regressive morphogenesis, although the frequent discrepancies between individuals of similar size implicate developmental age more than size in this effect. Collectively, these two effects provide only equivocal support for the hypothesis that direct development in plethodontids evolved via precocious TH activity. However, the unexpected transition from ceratobranchial replacement to ceratobranchial shortening in medium-sized larvae suggests that the former pathway requires a longer period of cell specification at low TH. Since ancestral plethodontids appear to have been distinguished by an exceptionally long larval period with exceptionally low TH activity, this developmental prerequisite may in turn be partly responsible for their singular evolution of ceratobranchial replacement. © 1995 Wiley-Liss, Inc.     

Energetics of metamorphic climax in the pickerel frog (Lithobates palustris)

Anuran metamorphosis, the transition from aquatic larvae to terrestrial juveniles, is accompanied by significant morphological, physiological, and behavioral changes. Timing of metamorphosis and final size, which can influence adult fitness, may depend on sufficient energy accumulated during the larval period to support metamorphosis. However, only two species of anurans have been examined for energetic costs of metamorphosis, Rana tigrina and Anaxyrus terrestris. Based on these species, it has been hypothesized that differences in energy expenditure are related to duration of metamorphosis. To compare energetic costs of metamorphosis among species and examine this hypothesis, we quantified the total energy required for metamorphosis of Lithobates palustris tadpoles by measuring oxygen consumption rates over the duration of metamorphic climax using closed-circuit respirometry. Total energy costs for L. palustris were positively related to tadpole mass and duration of metamorphic climax. However, larger tadpoles completed metamorphosis more efficiently because they used proportionally less total energy for metamorphic climax than smaller counterparts. Costs were intermediate to R. tigrina, a larger species with similar metamorphic duration, and A. terrestris, a smaller species with shorter metamorphic climax. The results supported the hypothesis that amphibian species with more slowly developing tadpoles, such as ranids, require more absolute energy for metamorphosis in comparison to more rapidly developing species like bufonids.     

Metamorphic Competence, a Major Adaptive Convergence in Marine Invertebrate Larvae

Larvae from diverse marine-invertebrate phyla are able to respondrapidly to environmental cues to settlement and to undergo veryrapid metamorphic morphogenesis because they share the developmentaltrait of metamorphic competence. The competent state, characteristicof larvae as diverse as those of cnidarian planulae, molluscanveligers, and barnacle cyprids, is one in which nearly all requisitejuvenile characters are present in the larva prior to settlement.Thus metamorphosis, in response to more or less specific environmentalcues (inducers), is mainly restricted to loss of larva-specificstructures and physiological processes. Competent larvae oftwo "model marine invertebrates" studied in the authors' laboratory,the serpulid polychaete Hydroides elegans and the nudibranchPhestilla sibogae, complete metamorphosis in about 12 and 20hr, respectively. Furthermore, little or no de novo gene actionappears to be required during the metamorphic induction processin these species. Contrasting greatly with the slow, hormonallyregulated metamorphic transitions of vertebrates and insects,competence and consequent rapid metamorphosis in marine invertebratelarvae are conjectured to have arisen in diverse phylogeneticclades because they allow larvae to continue to swim and feedin the planktonic realm while simultaneously permitting extremelyfast morphological transition from larval locomotory and feedingmodes to a different set of such modes that are adaptive tolife on the sea bottom.     

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide,Irgarol 1051

Abstract

The impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

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.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10?? M epinephrine. The concentration that inhibited metamorphosis by 50% (IC??) for chlorpromazine and amitriptyline was 1.6 x 10?? M and 6.6 x 10?? M, respectively. Idazoxan was less effective with an IC?? of 4.4 x 1013 M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K+ was not inhibited by 10?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Timing is everything: the effects of putative dopamine antagonists on metamorphosis vary with larval age and experimental duration in the prosobranch gastropod Crepidula fornicata

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 microM) 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 D(1) 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 D(2) 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(+).     

Predicting larval metamorphosis of Pacific lamprey Entosphenus tridentatus through measurements of length and mass

The utility of length and mass measurements to predict the larval metamorphosis of Pacific lamprey Entosphenus tridentatus was evaluated. During 2004–2008, larval E. tridentatus were collected from Cedar Creek (Washington, USA) in either the spring or autumn, measured for total length and total mass, reared in captivity and monitored for metamorphosis. The minimum total length, total mass and condition factor of larvae that were observed to go through metamorphosis were 102 mm, 2.0 g and 1.52, respectively. Logistic models indicated that total length and condition factor in both spring and autumn were the most significant variables for predicting metamorphosis of Pacific lamprey during the subsequent summer. Mass in the autumn also appeared important to predict whether metamorphosis occurred in the subsequent summer. Collectively, all models using specific minimums of total length, total mass or condition factor of larvae as criteria for them to metamorphose were sometimes (5 of 14 cases) able to predict the percentage of larvae that would metamorphose but rarely (1 of 12 cases) able to predict which individual larvae would metamorphose. Similar to other anadromous species of lampreys, the size and condition of larval E. tridentatus have utility for predicting metamorphic fate.     

Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867)

Jungblut, L.D., Pozzi, A.G. and Paz, D.A. 2010. Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867). — Acta Zoologica (Stockholm) 92 : 305–315. The olfactory and the vomeronasal system are the two major chemosensory systems found in terrestrial vertebrates. Among tetrapods, amphibians are unique in having an aquatic larval stage, followed by metamorphosis to a terrestrial adult. In the present work, we studied the histological development of the olfactory and vomeronasal organ and associated multicellular glands of the toad Rhinella (Bufo) arenarum, from early poshatching larva to postmetamorphic toadlets. As in other bufonids, the olfactory epithelium of R. arenarum in larvae is divided into dorsal and ventral branches in the rostral and mid‐nasal regions. At metamorphic climax, the larval pattern changes drastically and the adult olfactory configuration develops. Bowman’s glands appear in the olfactory epithelium of R. arenarum at the onset of metamorphic climax. The vomeronasal epithelium develops early in larval development in R. arenarum, around the time of operculum development. Interestingly, a novel sensory epithelium develops in the floor of the principal chamber of R. arenarum at metamorphic climax. This novel sensory epithelium resembles larval sensory epithelium lacking Bowman’s glands, and suggests that these animals would be able to sense not only air‐borne, but also water‐borne odors during their adult terrestrial life.     

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis

 The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T₃ action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T₃ and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59–60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T₃ (10^-8 M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occured specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T₃ action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T₃ and 32 times as much by the action of T₃ plus HC, indicating the important action of T₃–HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T₃–HC synergism on adult erythroblast proliferation and T₃-mediated programmed death of larval precursor cells. Accepted: 14 January 1999     

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.     

Induction of larval settlement and metamorphosis in the donkey-ear abalone,Haliotis asinina Linnaeus,by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.