Echinus Rudiment Research Articles

Initial report of \u03b3-aminobutyric acidergic locomotion regulatory system and its 3-mercaptopropionic acid-sensitivity in metamorphic juvenile of sea urchin, Hemicentrotus pulcherrimus

The γ-aminobutyric acid (GABA) signal transmission system (GSTS) contributes to larval swimming through the regulation of ciliary beating. However, whether this system also contributes to the primary podia (PP)-generated motility of juveniles remained unclear. The present study aimed to elucidate the involvement of the GSTS in the motility of metamorphic juveniles (juveniles) (1) by immunohistochemically elucidating the location of molecular constituents of the PP, and (2) by inhibiting the activity of GΑΒΑ decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). During metamorphosis, the echinus rudiment protrudes its PP out of the body surface in 8-arm plutei. The PP expresses immunopositive signal (-IS) of GAD, GABA, GABAA receptor and tropomyosin, and is constituted with the GABA-IS negative distal tip and the GABA/GAD-IS gaiter region. The latter radiates distal projections to the disc that contains a GAD-IS cellular network. The juvenile body cavity houses a GABA/βIII-tubulin-IS Penta-radial ring (PrR) that extends branches into each PP and several bridges to the GAD/GABA-IS Penta-radial plate (PrP) on the oral side but does not reach to the gaiter region. 3-MPA reversibly inhibits the juvenile motility and GABA-IS expression in the PrR/PrP complex. This indicates that the complex is the major contributor to the GABAergic motility in juveniles.

Synergistic negative effects of thermal stress and altered food resources on echinoid larvae

Multiple changes to the marine environment under climate change can have additive or interactive (antagonistic or synergistic) effects on marine organisms. Prompted by observations of anomalously warm sea temperatures and low chlorophyll concentrations during the 2013–2016 warm “Blob” event in the Northeast Pacific Ocean, we examined the combined effects of thermal stress and a shift in food resources on the development of a larval echinoid (Strongylocentrotus droebachiensis) in the laboratory. A high concentration of phytoplankton yielded faster echinus rudiment development at warm versus historical temperature, indicating a mitigating effect of abundant food on thermal stress; however, low phytoplankton concentration or a shift in diet to suspended kelp detritus, yielded slow development and high mortality at warm temperature. The results indicate a synergistic negative effect of thermal stress and altered food resources on larvae of a keystone marine species.

A newly identified left-right asymmetry in larval sea urchins.

Directional asymmetry (DA) in body form is a widespread phenomenon in animals and plants alike, and a functional understanding of such asymmetries can offer insights into the ways in which ecology and development interface to drive evolution. Echinoids (sea urchins, sand dollars and their kin) with planktotrophic development have a bilaterally symmetrical feeding pluteus larva that undergoes a dramatic metamorphosis into a pentameral juvenile that enters the benthos at settlement. The earliest stage of this transformation involves a DA: a left-side invagination in mid-stage larvae leads to the formation of the oral field of the juvenile via a directionally asymmetric structure called the echinus rudiment. Here, we show for the first time in two echinoid species that there is a corresponding DA in the overall shape of the larva: late-stage plutei have consistently shorter arms specifically on the rudiment (left) side. We then demonstrate a mechanistic connection between the rudiment and arm length asymmetries by examining rare, anomalous purple urchin larvae that have rudiments on both the left and the right side. Our data suggest that this asymmetry is probably a broadly shared feature characterizing ontogeny in the class Echinoidea. We propose several functional hypotheses—including developmental constraints and water column stability—to account for this newly identified asymmetry.

Mechanism maintaining dense beds of the sand dollar Scaphechinus mirabilis in northern Japan

The sand dollar Scaphechinus mirabilis, which inhabits the intertidal and subtidal sandy bottom in the Japan Sea and northwest Pacific Ocean near Japan, forms dense beds at some places for several decades. I investigated how this sand dollar maintains long-lasting dense beds by studying the population dynamics over 16 months in a dense bed dominated by adults with a test length of about 40 mm. The density and size structure of the population hardly varied throughout the investigation. The sand dollars reproduced in November, but no juveniles were recruited in the dense bed. In the laboratory, plutei with a well-developed echinus rudiment metamorphosed faster in sand in which an adult was kept for 2 days. This suggests that plutei selectively settle in and around existing habitats. On the other hand, most juveniles died when they were kept for 3 months with 20 adults whose density was equal to that of the dense bed. In contrast, few juveniles died when they were kept with 20 dead tests, or 10 dead tests and 10 adults. Most juveniles died in vessels in which the sand was shaken continuously to simulate bioturbation by adults. I investigated the size structure of the population in 8 habitats. Juveniles were abundant in habitats with few adults, but scarce in habitats with dense adults. This result indicates that the plutei settle in and around existing habitats, but that the bioturbation caused by adults kills most settled juveniles. It seems that the dense beds are maintained by the gradual immigration of juveniles that are able to settle and grow around there, or by the occupancy of juveniles after the mass mortality of adults.

NO/cGMP Signaling and HSP90 Activity Represses Metamorphosis in the Sea UrchinLytechinus pictus

Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod. We present evidence for a similar role in the sea urchin Lytechinus pictus. NO commonly signals via soluble guanylyl cyclase (sGC). Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in echinoid larvae as well. Pluteus larvae containing juvenile rudiments were treated with either radicicol L- or D-nitroarginine-methyl-ester (L-NAME and D-NAME), or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitors of HSP90, NOS, and sGC, respectively. In all instances, drug treatment significantly increased the frequency of metamorphosis. SNAP, a NO donor, suppressed the inductive properties of L-NAME and biofilm, a natural inducer of metamorphosis. NADPH diaphorase histochemistry indicated NOS activity in cells in the lower lip of the larval mouth, the preoral hood, the gut, and in the tube feet of the echinus rudiment. Histochemical staining coincided with NOS immunostaining. Microsurgical removal of the oral hood or the pre-oral hood did not induce metamorphosis, but larvae lacking these structures retained the capacity to metamorphose in response to ODQ. We propose that the production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduces the production of NO, and consequently cGMP, to initiate metamorphosis.

Effects of delayed metamorphosis and food rations on the perimetamorphic events in the echinoid Paracentrotus lividus (Lamarck, 1816) (Echinodermata)

Effect of delayed metamorphosis and food ration on late (competent) larvae and postlarvae of Paracentrotus lividus were investigated. Metamorphosis of competent larvae was either not delayed or delayed from 1 up to 4 days. Larvae were starved or submitted to two different food rations of the algal species Phaeodactylum tricornutum. Larvae during the prolonged competence period and the resulting postlarvae were characterised by: (1) the size of the larval body, (2) the size of the rudiment, (3) the rate of metamorphosis, (4) the size of postlarvae 24 h after metamorphosis, (5) the rate of opening of mouth and anus, (6) the rate of survival, and (7) the growth rate of early postmetamorphic individuals. Both the width of the larval body and the diameter of the echinus rudiment grew in competent larvae that were fed. Unfed larvae did not grow. There was no significant difference in growth between the two food rations. The rate of metamorphosis was higher with larvae that metamorphosed soon after they became competent. Lower capacity of larvae to metamorphose during the delay period was associated with treatments yielding a greater larval width and rudiment diameter during the same period. Postlarval development was affected by a delayed metamorphosis treatment inflicted on competent larvae before metamorphosis. Acquisition of exotrophy happened earlier in postlarvae issued from prolonged competent larvae whatever the larval food rations. The delay treatment negatively affected the development of the digestive tract though it positively affected the growth of early postmetamorphic individuals during the first 6 days following metamorphosis. However, selective mortality occurred afterwards as bigger individuals died preferentially.

Cellular analyses of sea urchin metamorphosis

Larvae of marine invertebrates undergo metamorphosis in response to environmental cues (Chia & Burke, 1978). In sea urchins, free fatty acids (Kitamura et al., 1993), dibromomethane (Taniguchi et al., 1994), pheromonal peptides (Burke, 1984) and L-glutamine (Yazaki & Harashima, 1994; Yazaki, 1995) have been known as metamorphosis-inducing substances. The mechanisms by which cells respond to these cues and how the larval tissues are absorbed have not been clear, however. In the present study, we used L-glutamine (Gln) and a natural cue, green algae (Ulvella sp.), to induce metamorphosis of Hemicentrotus pulcherrimus and Anthocidaris crassispina, and investigated the intracellular changes during metamorphosis.After being subjected to 10−5–10−3 M Gln for 10–24 h, larvae cease swimming, settle, begin to retract their larval arms, extrude the primary podia and finally evert their echinus rudiment (ER). In H. pulcherrimus, larvae retracted their arms from 6 h to 24 h after the start of Gln treatment and then everted the ER. A. crassispina larvae underwent similar processes to those of H. pulcherrimus. The larval surface is composed of squamous epithelium and columnar epithelium. The epithelium of the ciliary bands or epaulets is columnar.In the squamous epithelium, the nuclear chromatin in the larval arms and body, and in the oesophagus, markedly condensed after treatment with Gln for 24 h. Electron microscopy revealed swelling of both nuclei and mitochondria, while their membranes seemed to be intact. In the cytoplasm, lipid-like structures and electron-dense substances appeared. A further 24 h after Gln treatment, the chromatin condensation had progressed. Most nuclei in which chromatin had condensed were positive to the TUNEL assay, which detects DNA fragmentation. These results suggest that cell death in the squamous epithelium is apoptotic rather than necrotic.

Effect of microalgae treated with natural toxins on the nutrition and development of filter-feeding sea-urchin larvae

Filter-feeding larvae of the sea urchin Paracentrotus lividus fed phytoplankton previously exposed to sublethal amounts of toxins were chosen as a model to assess the potential effects of natural toxins on survivorship, development and feeding behaviour. The marine microalga Cricosphaera elongata was incubated with organic extract of the green seaweed Caulerpa taxifolia and then offered to larvae at different stages of development. Sensitivity to the toxin-treated food depended on the larval stage at which exposure began. Larvae reared from first feeding (4-arm stage) with toxin-treated microalgae were most sensitive (25% survival, delay in development and a metamorphosis rate of 32%). A diet begun at the 8-arm stage caused a decrease in survival and abnormal development; however, all the remaining larvae achieved metamorphosis. Certain ontogenic stages were more sensitive than others; in all treatments, mortality rate was highest during the formation of the echinus rudiment. Feeding experiments comparing ingestion of inert and toxin-treated polystyrene microspheres suggested that larvae discriminate against toxin-treated particles.

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres.

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.

Quantitative analysis of metamorphosis induced by L-glutamine in embryos of the sea urchin, Hemicentrotus pulcherrimus.

Metamorphosis of the sea urchin, Hemicentrotus pulcherrimus, can be induced by L-glutamine as reported previously for Pseudocentrotus depressus [15]. To analyze more precisely the process of metamorphosis induced by L-glutamine, the development of the echinus rudiment (ER) was classified into six stages. The stage at which the larvae underwent the normal metamorphosis by glutamine treatment was confirmed. The time of the glutamine treatment required for metamorphosis (the eversion of ER) was over 10 hr but treatment for more than 25 hr tended to decrease the number of metamorphosed larvae, although the larval arms had mostly been resorbed. The time of glutamine treatment to induce the metamorphosis, depended on the development of ER; more time was required for the younger larvae and less time for the older. The high mitotic activities observed in the cells of the ciliary bands were markedly decreased in the glutamine-treated larvae to metamorphose. These findings suggested that a degenerative process of metamorphosis including cell death is induced by L-glutamine.

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma.

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.

The establishment of bilateral asymmetry in sea urchin embryos

ABSTRACT Although much is known about the specification and determination of the two primary axes (animal/vegetal and dorsoventral or oral/aboral) in a number of embryos, little is understood about bilaterality. In the sea urchin, left/right asymmetry is crucial to normal development as the echinus or adult rudiment is positioned on the left side of the larva. We examined the establishment of bilateral asymmetry in Lytechnis variegatus embryos by determining the relationship of the first cleavage planes to the left/right axis. Embryos were bisected at different times to determine when the bilateral axis is committed. These lineage tracing and cell separation experiments demonstrated that the first cleavage plane divides the embryo into left and right halves, although this is conditional until after late blastula stage. The relationship between the specification of the dorsoventral axis and the bilateral axis was examined experimentally. In other species when the dorsal and ventral halves of early echinoderm embryos (preblastula) are separated, the dorsal half often reverses (180°) its dorsoventral axis. We asked whether those larvae with an inverted dorsoventral axis would shift the position of the echinus rudiment from the original left side to the new left side. If so, it would demonstrate that the larval asymmetry is dependent upon specification of the dorsoventral axis. Using a combination of lineage tracing and cell separation techniques, we show that the left/right asymmetry is specified with respect to the dorsoventral axis.

Five tests of food‐limited growth of larvae in coastal waters by comparisons of rates of development and form of echinoplutei

Any single method of testing food limitation can be misleading or inconclusive. We therefore used five different tests for food‐limited growth of larvae of a sea urchin from the northwest Mediterranean: 1. In both spring and autumn, larvae given a nearly natural ration of food from daily changes of seawater grew and developed more slowly than larvae with the same water enhanced with a cultured alga. 2. Larvae given a nearly natural ration of food developed more slowly in autumn, when concentrations of chlorophyll a and particles in the plankton were lower, than in spring when these indications of food were higher. 3. Larvae given the enhanced ration grew and developed more rapidly than larvae in presumed cohorts in the plankton. 4. In both spring and autumn, larvae in the field had a food‐limited form (longer arms and delayed formation of the echinus rudiment) in contrast to larvae given an enhanced ration. 5. Larvae in the autumn plankton had a more food‐limited form than larvae in the spring plankton. Results of all five tests indicated food‐limited growth in coastal waters in autumn; three indicated food‐limited growth in spring. The concentrations of natural food were not unusually low. Food‐limited growth suggests that these larvae may commonly feed with maximal or near‐maximal clearance rates. If echinoid larval growth is limited by food under these conditions, it is likely that growth of other larval invertebrates is food limited in many coastal waters.

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchinHeliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis inH. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.

Heat waves, baby booms, and the destruction of kelp beds by sea urchins

Large populations of sea urchins, Strongylocentrotus droebachiensis (Muller), destroyed kelp beds along the Atlantic coast of Nova Scotia in the 1960's and 1970's. The origin of these large sea urchin populations is not understood. We have investigated the potential influence of variable growth and development of the planktonic larvae of sea urchins (in response to temperature and food abundance) on recruitment of benthic juveniles. The adult sea urchins were collected at Sandy Cove, Digby County, Nova Scotia, Canada, in December 1986. Temperature strongly affected larval size and the growth of the echinus rudiment within the range 3° to 9°C, and larvae grew most rapidly at 14°C. Food abundance had a smaller effect on larval growth, and these effects were apparent only at high temperature. Larvae fed the same concentration of two different algal food species grew and developed similarly. Correspondence between spring temperature variation and qualitative variation in sea urchin recruitment, as well as strong temperature effects on larval growth in culture, and the occurrence of a large, positive temperature anomaly in June 1960, all suggest that temperature effects on larval growth and development may have led to intense sea urchin recruitment in 1960 and the appearance of large adult populations 4 to 6 yr later. This result invites further research.

Comparative morphometrics of echinoderm larvae. II. Larval size, shape, growth, and the scaling of feeding and metabolism in echinoplutei

The relationships among egg size, larval growth and form, and the functional consequences of larval form, (e.g. the changing relationship between ciliated band length and metabolic activity) were compared among four co-occurring echinoids, Strongylocentrotus droebachiensis (O. F. Müller), S. franciscanus (A. Agassiz), S. purpuratus (Stimpson), and Dendraster excentricus (Eschscholtz). Development time from fertilization to the formation of the echinus rudiment, just prior to metamorphic competence, was inversely related to egg volume. Egg sizes spanned a 6-fold range in volume, corresponding to a doubling of developmental duration. Species with smaller eggs should have greater fecundity per unit reproductive effort. However, the larval stages should suffer greater planktonic predation as a result of slower development. In general, the initial differences in larval size and shape that are related to egg size do not persist throughout development. The quantitative importance of shape change in increasing the size of feeding structures relative to support structures is uniformly high (80–85% of total change is allometric) in these species. During the period of arm formation (2-armed stage to 8-armed stage), ciliated band length scaled with strong positive allometry with respect to body length. The allometric exponents ranged from 2.7 to 3.4 and were positively related to egg size. Species with larger eggs have greater inherent capability for increasing the size of feeding structures relative to support structures during growth. Likewise, the sealing of feeding capability with respect to metabolic activity was positively related to egg size, with the allometric exponents ranging from 0.8 to 1.5. An important consequence of increased egg size is the increased ability to seale feeding structures positively with respect to the geometric and energetic size of the larva.

Food limited growth and development of larvae: Experiments with natural sea water

Food limitation of growth and development of larvae in coastal waters was tested with larvae of five species of marine invertebrates from the San Juan Islands, WA, U.S.A. The larvae were fed three rations: food in natural sea water, food in natural sea water with cultured algae added, or food in natural sea water with the particle concentration reduced one-fifth by addition of membrane filtered sea water. Larvae of four of the five species grew significantly faster with the enhanced ration than with natural sea water. Larvae of the sand dollar Dendraster excentricus (Eschscholtz) metamorphosed on the 21st day after fertilization with the enhanced ration, on the 27th day with the unaltered ration, and on the 47th day with the reduced ration. The size of the echinus rudiment at metamorphosis of D. excentricus was independent of food concentrations. Since the concentration of chlorophyll a in the natural sea water of this study is common in coastal waters, natural food supplies may commonly limit growth and development of larvae.

Morphometric and metabolic analysis of the growth and form of an echinopluteus

The pluteus larva of the echinoid, Dendraster excentricus (Eschscholtz, 1829), increases in size and changes shape during development. Larval length increases from 311 to 699 μm between the two-armed stage and the formation of the echinus rudiment. Protein content increases from 130 to 600 ng, while metabolic activity increases 28-fold during this period. Larval volume increases from 1867 × l0 3μm 3 to 5506 × 10 3μxm 3 during growth from the two- to the eight-armed stage. Initially, shape changes through the formation of four pairs of larval arms. Later, body proportions are altered because of positive allometric growth of the posterodorsal and postoral arms. The changes in body form from the two- to the eight-armed stage result in positive scaling of larval surface area with respect to body volume ( b = 1.6, size independent) ( b is the allometric coefficient, Y= aX b ). The larval feeding structure (ciliated band) grows with positive allometry with respect to midline body length ( b = 2.8), overall larval length ( b = 2.1), surface area ( b = 1.3), and volume ( b = 1.4). Shape change, rather than size increase, accounts for most (83%) of the increase in feeding capability. Larval body wall tissue volume scales isometrically with metabolic demand ( b = 0.9) during the period of arm formation. Throughout development, most of the growth of the larva occurs in the arms. By the eight-armed stage the arms account for 74% of the ciliated band, 48% of the surface area, 27% of the total volume, and 79% of the body wall tissue volume. The contribution of the arms to these measures of area and volume increases continuously during larval development. In contrast, the percentage of the ciliated band on the arms increases from 41% in the two-armed pluteus to 64% in the four-armed stage and-then remains nearly constant throughout larval growth, averaging 68%. The important functional consequence of the changes in body form that characterize the pluteus growth pattern is the maintenance of feeding capability relative to larval body size (body wall tissue volume, b = 1.0) and metabolic demand ( b = 1.1). Because later larval stages can eat a wider size range of food organisms, the isometry of clearance capability and metabolism indicates that later stages should be less food limited.

The reproductive system, embryonic development, larval development and metamorphosis of the sea urchin Heliocidaris erythrogramma (Val.) (Echinoidea : Echinometridae)

H. erythrogramma has an annual reproductive cycle, spawning asynchronously. The female cycle has five phases: resting, regenerating, mature, spent and regressing; the male cycle omits the regression phase. Regression and resting occur only during April-August; regeneration begins in September; during December-March individual urchins pass through the mature, spent and regenerating phases two or three times. The egg averages 400 �m diameter and floats at the water surface. At 24-25�C, equal, total, radial cleavage yields a wrinkled coeloblastula by 6-7 hours; this undergoes egression, becoming a spherical stereoblastula by 13-14 hours; during this process an acellular central yolk mass is segregated from a peripheral columnar blastoderm. The ciliated stereoblastula gastrulates by a typical diphasic invagination, beginning at 15 hours, and the gastrulating embryo hatches as a ciliated, planktonic yolk-larva at 15-16 hours. Lecithotrophic development of the yolk-larva to a uniformly ciliated, planktonic vitellaria is complete by 1.5 days, and the larva metamorphoses gradually to a lecithotrophic juvenile over the next 3.5 days. It remains planktonic during the first half of this period (to 3.75 days), but settles during the second half (to 5 days). The juvenile begins to feed >3 weeks after settling. Vegetally proliferating mesenchyme invades the yolk mass during gastrulation. Invagination of the archenteron is followed by evagination of a primary pair of coelomic pouches from its wall; the left enterocoel forms the hydrocoel. Other coelomic cavities are re-formed by schizocoely. The larval gut rudiment temporarily loses connection with the exterior after the blastopore closes. The proctodaeum and stomodaeum of the juvenile form secondarily during and after metamorphosis. Ectodermal development of the echinus rudiment, though gradual, is typical of echinoids. The blastulation of H. evythrogramma is unique among large-egged echinoderms in combining egression of a wrinkled coeloblastula with segregation of a blastoderm around a central yolk mass. The development of the species supports the view that the vitellaria larva is secondarily evolved within the Echinoidea.

Experimentally-Induced Metamorphosis in Echinus

(1) Treatment of advanced larvae of Echinus miliaris with very dilute solutions of HgCl2 (about m/2 X 106 rapidly brings about precocious metamorphosis, a young echinus in one case surviving eight days in spite of the absence of food. (2) This appears to be caused through the differential susceptibility of larval tissues and echinus rudiment. The former are more affected by the poison, begin to dedifferentiate, and can then be readily resorbed by the echinus rudiment. (3) When the echinus rudiment is small, metamorphosis is less rapid and may be incomplete, both larval and echinus tissues being dedifferentiated. Even when metamorphosis in such cases is complete, it is often abnormal, the young echinus possessing small or no tube-feet or spines. Parallel abnormalities are to be found in artificial (thyroid-induced) metamorphosis in anuran tadpoles. (4) It is suggested that a similar mechanism is operative in the normal metamorphosis of echinids, the larval tissues dedifferentiating when the weight of the echinus rudiment causes the organism to sink away from the favorable food and oxygen conditions of the surface. (5) Some dedifferentiation phases are figured.