Actin-mediated retraction of the larval epidermis during metamorphosis of the sand dollar,Dendraster excentricus

Summary During the first 15 to 20 min of metamorphosis the larval arms are retracted and resorbed into the aboral surface of the juvenile. Arms excised from metamorphosing larvae will undergo a sequence of contraction and histolysis that is identical to that occurring in intact larvae. Prior to and during metamorphosis, epidermal cells contain bundles of 5 to 7-nm microfilaments in arrays radiating apically from the base of the cells. Sparse microfilaments also occur near the plasmalemma of epidermal cells and some mesenchymal cells in larvae fixed during metamorphosis. Contraction of excised arms is reversibly inhibited by treatment with cytochalasin B, and microfilaments bind myosin subfragment-l. Indirect immunofluorescence of larval arms using an antibody against chicken-muscle actin and staining with the F-actin specific probe, NDB phallacidin indicate that the arms contain actin distributed in a manner consistent with ultrastructural findings. It is proposed that retraction of the larval arms during metamorphosis is produced by an actin-mediated change in shape of the epidermal cells.     

Centriole behavior in chloramphenicol-treated eggs of the sand dollar,Dendraster excentricus

Summary This electron microscope study was undertaken to test the prediction made from an indirect assay method for mitotic centers (centrioles), that chloramphenicol inhibits centriole replication during first cleavage division in the eggs of the sand dollar,Dendraster excentricus. Extensive serial sectioning through both untreated and chloramphenicol-treated eggs, coupled with thorough examination of these sections, has demonstrated that 57% of the untreated eggs and 14% of the chloramphenicol-treated eggs contained paired centrioles at a time when centriole pairs normally exist. This study thus gives direct evidence that chloramphenicol inhibits centriole replication.     

Fertilization selection on egg and jelly-coat size in the sand dollar Dendraster excentricus

Organisms with external fertilization are often sperm limited, and in echinoids, larger eggs have a higher probability of fertilization than smaller eggs. This difference is thought to be a result of the more frequent sperm-egg collisions experienced by larger targets. Here we report how two components of egg target size, the egg cell and jelly coat, contributed to fertilization success in a selection experiment. We used a cross-sectional analysis of correlated characters to estimate the selection gradients on egg and jelly-coat size in five replicate male pairs of the sand dollar Dendraster excentricus. Results indicated that eggs with larger cells and jelly coats were preferentially fertilized under sperm limitation in the laboratory. The selection gradients were an average of 922% steeper for egg than for jelly-coat size. The standardized selection gradients for egg and jelly-coat size were similar. Our results suggest that fertilization selection can act on both egg-cell and jelly-coat size but that an increase in egg-cell volume is much more likely to increase fertilization success than an equal change in jelly-coat volume. The strengths of the selection gradients were inversely related to the correlation of egg traits across replicate egg clutches. This result suggests the importance of replication in studies of selection of correlated characters.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Effect of 3'-deoxyadenosine (cordycepin) on the early development of the sand dollar, Dendraster excentricus

The embryos of the sand dollar Dendraster excentricus have been examined with regard to their ability to undergo the early events of larval development in the presence of cordycepin, a reported inhibitor of RNA adenylylation. It has been shown that all the morphogenetic events from hatching to prism are inhibited by cordycepin at a concentration of 25 μg/ml, while the aspects of development prior to hatching (cleavage, blastulation, and the formation of cilia) are not affected by cordycepin. The period of sensitivity of each developmental event to cordycepin has been determined; for the early developmental processes this period substantially precedes the event, while for later processes there is a closer temporal association. In embryos treated with cordycepin, DNA synthesis and cleavage are unaffected prior to hatching, but subsequently are blocked, whereas respiration and total RNA synthesis per cell remain unaffected.     

Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar,Dendraster excentricus

Summary The pattern of appearance of ribosomes, newly synthesized mRNA, and poly(A)-containing mRNA in polyribosomes has been examined in sand dollar embryos. From early blastula until shortly before hatching small polyribosomes engaged in histone synthesis predominate. At the time of hatching, when the rate of cell increase is maximal, the proportion of poly(A)-containing RNA in polyribosomes is low. After hatching a new class of large polyribosomes appears and the amount of poly(A)-containing polyribosomal RNA increases. Cordycepin, an inhibitor of RNA adenylylation, prevents the appearance of the large polyribosomes after hatching as well as the increase in poly(A)-containing polyribosomal RNA.     

Histology of the pedicellariae of the Sand dollar, Dendraster excentricus (Echinodermata)

The pedicellariae in Dendraster excentricus are the bidentate type with a density of 80/cm². There are two distinct size groups; the larger ones measure 0.6 mm long while the small ones are only 0.14 mm. One interesting feature is the fluid of highly sulphated acid mucopolysaccharide in the lumen of the neck. This fluid functions as a hydroskeleton while the flexor muscle operates on it for complicated movement of the neck. The possibility that this fluid is also toxic and so acts as a defence mechanism is discussed. All the muscles involved in the movement of pedicellariae are smooth muscle fibres.     

Action of trypsin on the eggs of Dendraster excentricus

Crystalline trypsin in 3 × 10^?8 M concentration and higher, elicits fertilization membranes in the unfertilized eggs of Dendraster excentricus. These membranes are adequate in artificial parthenogenesis. If the action of trypsin on these eggs is continued for two or three hours the result is first, digestion of the membranes, followed later by reduction of the egg to amoeboid form. When fertilized, some of the partially digested eggs segment and form irregular cell masses, thus demonstrating that, in response to trypsin, there is first the cortical reaction giving rise to the fertilization membrane, and second, the progressive digestion and disintegration of the cytoplasm.Chymotrypsin causes rounding of the unfertilized eggs and, in rare instances, a few membranes, but the enzyme is not an adequate parthenogenetic agent.Fertilization of the egg renders the cytoplasm resistant to trypsin. The facts lead to the suggestion that fertilization liberates trypsin inhibitors in the cytoplasm.     

Pigmented Follicle Cells and the Maturation of Oocytes in the Sand Dollar, Dendraster excentricus

Early in the spawning season female D. excentricus can be induced to spawn oocytes in the late stages of oogenesis. Observations with light microscopy indicate that pigment cells migrate in the jelly coat away from the surface of oocytes coincident with the final growth of the oocyte and its maturation. The pigment cells undergo a series of changes in shape and the oocyte elaborates arrays of long microvilli as the cells elevate from the surface of the oocyte. Transmission electron microscopy (TEM) observations of oocytes fixed within the ovary indicate that features of the sequence observed in the spawned oocytes normally take place within the ovary, prior to spawning. Extracts of the pigment cells induce a low level of germinal vesicle breakdown in asteroid oocytes. It is proposed that these cells are homologous to the follicle cells of other echinoderms and are involved in stimulating the maturation of oocytes.     

Biomechanics of Larval Morphology Affect Swimming: Insights from the Sand Dollars Dendraster excentricus

Most planktonic larvae of marine invertebrates are denser than sea water, and rely on swimming to locate food, navigate advective currents, and avoid predators. Therefore, swimming behaviors play important roles in larval survival and dispersal. Larval bodies are often complex and highly variable across developmental stages and environmental conditions. These complex morphologies reflect compromises among multiple evolutionary pressures, including maintaining the ability to swim. Here, I highlight metrics of swimming performance, their relationships with morphology, and the roles of behavior in modulating larval swimming within biomechanical limits. Sand dollars have a representative larval morphology using long ciliated projections for swimming and feeding. Observed larval sand dollars fell within a narrow range of key morphological parameters that maximized their abilities to maintain directed upward movement over the most diverse flow fields, outperforming hypothetical alternatives in a numerical model. Ontogenetic changes in larval morphology also led to different vertical movements in simulated flow fields, implying stage-dependent vertical distributions and lateral transport. These model outcomes suggest a tight coupling between larval morphology and swimming. Environmental stressors, such as changes in temperature and pH, can therefore affect larval swimming through short-term behavioral adjustments and long-term changes in morphology. Larval sand dollars reared under elevated pCO(2) conditions had significantly different morphology, but not swimming speeds or trajectories. Geometric morphometric analysis showed a pH-dependent, size-mediated change in shape, suggesting a coordinated morphological adjustment to maintain swimming performance under acidified conditions. Quantification of the biomechanics and behavioral aspects of swimming improves predictions of larval survival and dispersal under present-day and future environmental conditions.     

Localization of a SALMFamide Neuropeptide in the Larval Nervous System of the Sand Dollar Dendraster excentricus

Using immunocytochemical methods we describe the localization of serotonin and the SALMFamide peptide, S1 (GFNSALMFamide), during embryonic and larval development of the echinoid Dendraster excentricus. Anti-SI immunoreactivity first appears in the apical ganglion in late gastrulae at the same time as anti-serotonin immunoreactivity. Initially, anti-S1 immunoreactivity is restricted to fibres of the neuropile, but in later feeding stages, cell bodies are also immunoreactive. Anti-S1 immunoreactivity appears as 2–4 cells in the oral ganglion of early prism stage larvae, whereas anti-serotonin immunoreactivity does not occur in the oral ganglion until the 8-arm stage. Anti-S1 immunoreactivity also occurs in diffuse fibres in the oesophagus and in a single fibre encircling the pyloric sphincter of the gut. A reticular network associated with the apical surface of the epithelial cells of the vestibule of the adult rudiment was anti-S1 immunoreactive. In double-labelling experiments, anti-serotonin and anti-S1 immunoreactivity co-localize in the neuropile of the apical ganglion. The distribution of S1, in association with putative sensory cells in the apical and oral ganglia and with muscles of the oesophagus and gut, suggests S1 may have diverse functions in the larval nervous system. The distribution of anti-S1 immunoreactivity in echinoid embryos and larvae supports the proposal that SALMFamide-like peptides are widely shared in echinoderms and potentially have a fundamental role in neural function.     

Growth, development and condition of Dendraster excentricus (Eschscholtz) larvae reared on natural and laboratory diets

Feeding invertebrate larvae may be food limited while developingin the ocean. If they are, then their time in the plankton isprolonged, which likely increases mortality. Food limitationcould be due to the quantity and/or quality of the food available.In an effort to answer how food type influences larval nutrition,we compared growth, development and lipid deposition for Dendrasterexcentricus larvae reared in natural seawater from two depths(1 and 20 m) and in filtered seawater on a monoculture laboratorydiet of 6 cells µL^–1 of the green alga Dunaliellatertiolecta (Butcher). Five days post-fertilization, larvaereared on the laboratory diet had developed to the latest stage,were the largest and had lipid deposits. Larvae reared on naturalsurface water were intermediate in size and developmental stage,and larvae reared in the water from 20 m depth were the smallestand developed the slowest. This trend continued at 8 days post-fertilizationwhen surface water diet larvae were similar in size to laboratorydiet larvae, but their juvenile rudiments were significantlysmaller. To assess food availability in each food treatment,we compared the concentration of chlorophyll (Chl) a, b andc in natural seawater from each depth and in D. tertiolectaculture in filtered seawater. Natural seawater collected fromthe surface had the highest concentration of Chl a and c, whereasChl b was not significantly different between treatments. IncreasedChl concentrations in the surface water are likely due to higherconcentrations of diatoms and dinoflagellates, which are typicallynot high-quality food items for echinoid larvae. Our resultssupport a hypothesis that echinoid larvae in the water columnmay be limited by food quality.     

Structure of the digestive tract of the pluteus larva of Dendraster excentricus (Echinodermata: Echinoida)

Summary Histological and ultrastructural observations of the digestive tract of eight-armed plutei of Dendraster excentricus are reported. The esophagus is divided into two regions. The uppermost is a narrow tube comprised of ciliated cells that assist in transporting food to the more bulbous lower esophagus where food particles are formed into a bolus prior to entering the stomach. The esophagus is surrounded by a network of smooth muscle fibers that are predominantly oriented circumferentially in the upper esophagus, and longitudinally in the lower esophagus. The musculature of the upper esophagus produces peristaltic contractions, whereas contractions of the muscle of the lower esophagus open the cardiac sphincter and force food from the lower esophagus into the stomach. Axons are associated with the ciliated cells and the muscles of the upper esophagus. The cardiac sphincter consists of a ring of myoepithelium, with cross-striated myofibrils oriented around the bases of the cells. The gastric epithelium is comprised of two cell types. Type I cells, which predominate, absorb and store nutrients, and may be the source of secreted digestive enzymes. Type II cells apparently phagocytize and intracellularly digest whole algal cells. The intestine is comprised of relatively unspecialized cells and probably functions primarily as a conductive tube for the elimination of undigested materials.     

Ontogeny of the holothurian larval nervous system: evolution of larval forms

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Peptidomics of the larval Drosophila melanogaster central nervous system

Neuropeptides regulate most, if not all, biological processes in the animal kingdom, but only seven have been isolated and sequenced from Drosophila melanogaster. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomics approach aims at the simultaneous identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their posttranslational modifications. Using nanoscale liquid chromatography combined with tandem mass spectrometry and data base mining, we analyzed the peptidome of the larval Drosophila central nervous system at the amino acid sequence level. We were able to provide biochemical evidence for the presence of 28 neuropeptides using an extract of only 50 larval Drosophila central nervous systems. Eighteen of these peptides are encoded in previously cloned or annotated precursor genes, although not all of them were predicted correctly. Eleven of these peptides were never purified before. Eight other peptides are entirely novel and are encoded in five different, not yet annotated genes. This neuropeptide expression profiling study also opens perspectives for other eukaryotic model systems, for which genome projects are completed or in progress.     

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2-cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10-15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.