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(+).     

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.     

Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus)

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.     

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

The effect of copper ions on behavior of larvae of the sea urchin Strongylocentrotus intermedius at different temperatures of sea water

We investigated the behavior of larvae of the sea urchin Strongylocentrotus intermedius at the blastula and early pluteus stages in the water column at a temperature of 20, 22, and 23.5°C and in the presence of copper in concentrations of 0.01 and 0.02 mg/l. In clean sea water, at all tested temperatures, sea urchin larvae preferred the subsurface layer to where they rose from the bottom of the vessel to form dense aggregations. In water supplemented with copper, the behavior of larvae at 20 and 22°C was not different from that in clean water. An increase in temperature to 23.5°C and the addition of copper altered the behavior of the larvae; they did not leave near-bottom water, did not rise to the surface, although they continued moving.     

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.     

Using KCl to determine size at competence for larvae of the marine gastropod Crepidula fornicata (L.)

Competent larvae of the marine gastropod Crepidula fornicata (L.) were induced to metamorphose (i.e., lose the velum) by elevating sea-water [KCl] by 5–50 mM. The response was optimal at 15–20-mM elevations, at which 50% metamorphosis was obtained in <4 h. Larvae that did not metamorphose during brief exposures (1–5 h) to elevated [KCl] generally maintained the larval form following transfer to control sea water, suggesting that competent larvae must be continuously immersed in the test solutions for metamorphosis to occur. The smallest larvae to respond to elevated [KCl] had shell lengths of ≈700–800 μm, the range of shell lengths within which larvae of this species become responsive to natural inducers. All larvae >≈1125 μm shell length metamorphosed in response to increased [KCl]. Rearing temperature may affect the size at which larvae of this species become responsive to K⁺. CaCl₂ (20-mM concentration elevations), GABA (4×10⁻⁷, 4×10⁻⁶ M), and NaCl (10–20-mM concentration elevations) generally failed to trigger metamorphosis. Twenty-mM elevations of [RbCl] and [CsCl] induced 100% metamorphosis but the juveniles were immobile and died after several days. Elevating [KCl] appears to be a reliable way to assess competence and trigger metamorphosis in larvae of C. fornicata.     

Why and how marine-invertebrate larvae metamorphose so fast

It is argued that larviparous development has evolved at least eight times among extant animals. A 'need for speed hypothesis' is proposed to explain profound convergence on a pattern of small larvae and rapid metamorphosis across six marine invertebrate clades. Shared selection pressures include limits to larval size, the plankton-to-benthos transition, extreme hazards on the benthos, and the profound helplessness of metamorphosing animals. The adaptive mechanisms include: (1) development of juvenile structures in larvae before they are metamorphically competent; (2) external cues trigger metamorphosis; and (3) rapid cell-to-cell conductance of the metamorphic signal to bring about rapid loss of larval structures and release of juvenile structures. Both pattern and mechanisms contrast in every regard with those of the other two major larviparous clades, Insecta and Amphibia.     

Acceleration of the Cleavage in Sea Urchin Eggs by Treatments with Local Anesthetics (II) Treatments with Some Other Local Anesthetics than Procaine*

Unfertilized sea urchin eggs were exposed to sea water solutions of local anesthetics, such as caffeine, tetracaine and ethyl urethane, and the herbicide, isopropyl N-phenyl carbamate (IPC) for 10min and returned to normal sea water. Then they were inseminated 5min later. When eggs were pre-treated with 1–2 mM caffeine, 0.02–0.05 mM tetracaine, 50–100 mM ethyl urethane and 2% saturated sea water of IPC, respectively, they could cleave and hatch earlier than the control eggs. However, when fertilized eggs were continuously post-treated with solutions of the agents except IPC at the same concentrations as those in the case of the pre-treatments, the fertilized eggs could not cleave or were retarded in development. The possible mechanisms of the cleavage acceleration by pre-treatments with local anesthetics were discussed.     

Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sibogae

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a factor from their adult prey, the coral Porites compressa. Levels of endogenous catecholamines increase 6 to 9 days after fertilization, when larvae become competent for metamorphosis. Six- to nine-day larvae, treated with the catecholamine precursor L-DOPA (0.01 mM for 0.5 h), were assayed for metamorphosis in response to coral inducer and for catecholamine content by high-performance liquid chromatography. L-DOPA treatment caused 20- to 50-fold increases in dopamine, with proportionally greater increases in younger larvae, so that L-DOPA-treated larvae of all ages contained similar levels of dopamine. A much smaller (about twofold) increase in norepinephrine occurred in all larvae. The treatment significantly potentiated the frequency of metamorphosis of 7- to 9-d larvae at low concentrations of inducer. In addition, L-DOPA treatment at 9 d increased aldehyde-induced fluorescence in cells that were also labeled in the controls, and revealed additional cells. However, all labeled cells were consistent with the locations of cells showing tyrosine-hydroxylase-like immunoreactivity. Catecholamines are likely to modulate metamorphosis in P. sibogae, but rising levels of catecholamines around the time of competence are insufficient alone to account for sensitivity to inducer in competent larvae.     

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.     

Laboratory culture of the larvae of Conus textile Linné (Gastropoda: Toxoglossa)

The larvae of the Indo-Pacific gastropod Conns textile Linné were reared in the laboratory from hatching through metamorphosis. Larvae fed a mixed phytoplankton culture of Isochrysis galbana and Phaeodactylum tricornutum grew at a rate of 0.06 mm/day and began metamorphosing 16 days after hatching. Unfed control cultures yielded no metamorphically competent larvae. Laboratory-reared larvae metamorphosed spontaneously on the walls of the fïberglass rearing tanks when their average shell length was 1.5 mm. Measurements made on field-collected Conns textile juveniles indicate that the larvae metamorphose at the same size in the laboratory as they do in nature.Rates of larval shell length increase and dry weight increase paralleled each other until metamorphosis. At this point, shell growth slowed while dry weight increased suddenly. It is suggested that this weight increase reflects calcification and strengthening of the fragile larval shell upon entering the benthic environment.     

Regulation of internal pH of sea urchin sperm. A role for the Na/K pump

In the absence of sodium, sea urchin sperm have an acidic internal pH. The addition of sodium, lithium, or ammonium, but not of potassium ions, induces an internal alkalization. If potassium is added in the presence of sodium, a further alkalization is obtained; in contrast, potassium addition in presence of Li+ or NH+4 does not change the internal pH. The K+-induced pHi change is inhibited by ouabain and when sperm are depleted of their ATP. A large part of the potassium influx is stimulated by Na+, but not Li+, and inhibited by ouabain and cellular ATP depletion. We conclude that activity of Na/K-ATPase pumps located in the plasma membrane of sea urchin sperm could play a role in regulating the internal pH of sea urchin sperm by recycling sodium ions that enter the cell through Na/H countermovements.     

Effects of adsorbed organic and primary fouling films on bryozoan settlement

Marine primary fouling films, which consist of molecular organic and microbial components, have been reported to facilitate colonization of immersed surfaces by marine fouling organisms. Larvae of the cosmopolitan fouling bryozoan Bugula neritina (Linnaeus) were offered various substrata for attachment and metamorphosis. The materials were offered (a) after detergent washing, (b) after sorption of dissolved organic molecular films, and (c) after formation of primary films consisting of both microbial and adsorbed organic material. Wettability of the substrata by sea water was determined by contact angle measurements for each substratum. On washed substrata, attachment was favored with contact angles greater than ≈45° (cos contact angle <0.7). Adsorbed surface films had no effect on the low settlement of larvae on glass and high settlement on plastics. Microbial primary films, however, made glass attractive and plastics unattractive. These settlement preference changes did not correlate with the changes in wettability observed on these substrata. Dispersion of larvae over the settlement surface was random except on wettable surfaces coated with bacterial films, where settlement was strongly clustered (contagious).     

Evolution of direct-developing larvae: selection vs loss

Observations of a sea urchin larvae show that most species adopt one of two life history strategies. One strategy is to make numerous small eggs, which develop into a larva with a required feeding period in the water column before metamorphosis. In contrast, the second strategy is to make fewer large eggs with a larva that does not feed, which reduces the time to metamorphosis and thus the time spent in the water column. The larvae associated with each strategy have distinct morphologies and developmental processes that reflect their feeding requirements, so that those that feed exhibit indirect development with a complex larva, and those that do not feed form a morphologically simplified larva and exhibit direct development. Phylogenetic studies show that, in sea urchins, a feeding larva, the pluteus, is the ancestral form and the morphologically simplified direct-developing larva is derived. The current hypothesis for evolution of the direct-developing larval form in sea urchins suggests that major developmental changes occur by neutral loss of larval features after the crucial transition to a nonfeeding life history strategy. We present evidence from Clypeaster rosaceus, a sea urchin with a life history intermediate to the two strategies, which indicates that major developmental changes for accelerated development have been selected for in a larva that can still feed and maintains an outward, pluteus morphology. We suggest that transformation of larval form has resulted from strong selection on early initiation and acceleration of adult development.     

The potential for cryopreserving larvae of the sea urchin, Evechinus chloroticus

Larvae of the sea urchin, Evechinus chloroticus, at varying stages of development, were assessed for their potential to survive cryopreservation. Ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), at concentrations of 1-2 M, were evaluated as cryoprotectants (CPAs) in freezing regimes initially based on methods established for freezing larvae of other sea urchin species. Subsequent work varied cooling rate, holding temperature, holding time, and plunge temperature. Ethylene glycol was less toxic to larvae than Me2SO. However, no larvae survived freezing and thawing in EG. Larvae frozen in Me2SO at the gastrula stage and 4-armed pluteus stage regained motility post-thawing. The most successful freezing regime cooled straws containing larvae in 1.5 M Me2SO from 0 to -35 degrees C at 2.5 degrees C min(-1), held at -35 degrees C for 5 min, then plunged straws into liquid nitrogen. Motility was high 2-4 h post-thawing using this regime but decreased markedly within 24 h. Some 4-armed pluteus larvae that survived beyond this time developed through to metamorphosis and settled. Different Me2SO concentrations and supplementary trehalose did not improve long-term survival. Large variation in post-thaw survival was observed among batches of larvae produced from different females.     

Alterations of the eyes during ontogenesis inAporrhais pespelecani (Mollusca,Caenogastropoda)

The cerebrally innervated eyes of metamorphically competent larvae, newly metamorphosed larvae, and adults ofAporrhais pespelecani are ultrastructurally investigated and compared. The eyes are composed of a lens, a cornea, and an everse retina. In adults, a humour is located behind the lens. The retina consists of two different types of cells: sensory cells and supportive cells. The present study confirms earlier results and demonstrates that the distal part of the sensory cells is altered during ontogenesis. In metamorphically competent larvae, the sensory cells are exclusively ciliary. In newly metamorphosed larvae and in adults, however, the sensory cells are of the mixed type, bearing both cilia and microvilli. Furthermore, the findings confirm that both the supportive and corneal cells, as well as the distal supportive cell processes which are restricted to the eyes of adults are involved in lens formation.     

Proteolytic processing of a sea urchin, ECM-localized protein into lower mol mass species possessing collagen-cleavage activity

The hyaline layer is an apically located extraembryonic matrix, which blankets the sea urchin embryo. Using gelatin substrate gel zymography, we have identified a number of gelatin-cleaving activities within the hyaline layer and defined a precursor-product processing pathway which leads to the appearance of 40- and 38-kDa activities coincident with the loss of a 50-kDa species. Proteolytic processing of the precursor required the presence of both CaCl2 and NaCl at concentrations similar to those found in sea water. The cleavage activities utilized both sea urchin and rat tail tendon gelatins as substrates but demonstrated a species-specific cleavage activity towards sea urchin collagen. The gelatin-cleaving activities were refractory to inhibition by 1,10-phenanthroline but were inhibited by benzamidine. This latter result defines the serine protease nature of the cleavage activities. Both the 40- and 38-kDa activities were found to comigrate with gelatin-cleaving activities present in the sea urchin embryo.     

Persistence and integration of cloned DNA in postembryonic sea urchins

Cloned DNA was injected into the cytoplasm of unfertilized sea urchin eggs which were then fertilized and cultured in the laboratory through metamorphosis. The exogenous DNA replicated manyfold and persisted for weeks in a majority of growing larvae, as shown by hydridizing “dot blots” of the DNA of single individuals with appropriate labeled probes. After metamorphosis 5–15% of the juvenile sea urchins retained the exogenous sequences. Genomic integration of the exogenous sequence was observed in the DNA of a postmetamorphosis juvenile.