Development of the hyaline layer around the planktonic embryos and larvae of the asteroid Patiriella calcar and the presence of associated bacteria

Summary

The hyaline layer (HL) around the embryos and larvae of Patiriella calcar is examined by transmission electron microscopy. P. calcar hatches at the gastrula stage and develops through a lecithotrophic planktonic brachiolaria. The hyaline layer of unhatched P. calcar is poorly developed and is comprised of wispy fibrils scattered among the epithelial microvilli. Fibrils are also occasionally seen associated with the inner surface of the fertilization envelope. By the hatched gastrula stage, the hyaline layer is organized into three strata: the intervillous layer, the supporting layer and the coarse outer meshwork layer. Seven-day-old brachiolaria also have a hyaline layer comprised of three strata. In these larvae the supporting layer elevates away from the epithelial surface due to the tuft-like organization of the underlying microvilli. This results in the formation of local outpockets giving the surface of the HL a lobed appearance. Bacteria are occassionally seen in the intervillous layer, particularly in association with the outpockets. These bacteria are phagocytosed by the epithelial cells and, in larvae that have bacteria, may play an augmentive role in larval nutrition. The structure of the hyaline layer of P. calcar is compared with that of the hyaline layer of other Patiriella species to determine if it is more similar to the external coats around its planktonic (P. regularis) or benthic (P. exigua) developing congeners. The comparison shows that the hyaline layer of P. calcar is virtually identical to that of P. regularis, a similarity that may reflect the pelagic life histories of these species.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.     

Lectin histochemistry of the hyaline layer around the larvae of Patiriella species (Asteroidea) with different developmental modes.

Larvae of sea stars are surrounded by an extracellular matrix called the hyaline layer. The lectin-binding properties of this matrix were investigated in an ultrastructural study of Patiriella species having different modes of development. The planktonic bipinnaria and brachiolaria of P. regularis and the planktonic brachiolaria of P. calcar demonstrated the same labeling of the hyaline layer for three lectins: Con A, SBA, and WGA. In both species the outer coarse meshwork stained for all three lectins, whereas the intervillous layer displayed patchy labeling. In the benthic brachiolaria of P. exigua, the outer coarse meshwork displayed heavy labeling for all three lectins. The heavy labeling of the outer coarse meshwork of P. exigua compared with that of the other species suggests an increased number of lectin binding sites in the hyaline layer of this species. The similar ultrastructure and histochemistry of the hyaline layer of P. regularis and P. calcar may reflect similar requirements of their extracellular cover in their planktonic environment. Lectin labeling shows that hypertrophy of the hyaline layer of P. exigua, in particular the outer coarse meshwork, involves elaboration of the carbohydrate composition of the matrix. Modifications seen in the ultrastructure and histochemistry of the hyaline layer of P. exigua appear to be associated with the evolution of benthic development.     

Ultrastructural aspects of the surface coatings of eggs and larvae of the starfish,Pisaster ochraceus,revealed by alcian blue

Eggs of the asteroid Pisaster ochraceus demonstrate cortical granules, a thick vitelline membrane, and a poorly stained jelly coat similar to that seen on the eggs of other echinoderms. When fixed in the presence of alcian blue the jelly coat is seen to be made up of three regions, an inner layer consisting of a meshwork of fibres, a middle layer of thicker fibres, and a dense outer layer. At fertilization the cortical granules release their contents into the potential space between the vitelline layers and a low fertilization membrane consisting of the vitelline layer and a dense component of the corticle granule is formed. Initially the remaining contents of the corticle granules form an amorphous hyaline layer that fills the space between the plasma membrane and the fertilization membrane. At hatching a distinct hyaline layer is present. It persists at least to the bipinnaria stage and consists of four distinct layers. A similar layer is also located over much of the early embryonic endoderm but is lost from the regions involved in the formation of the mesenchyme cells, coelom, and mouth just before these events take place. Numerous large clear vesicles are located in the apex of all cells associated with a hyaline layer. Where the hyaline layer is lacking, only scattered vesicles are present suggesting that the vesicles may be involved in maintenance of the layer. Attempts to identify elements of the hyaline layer by immunofluorescence demonstrated that it appears to bind both antisera and control sera in a nonspecific manner.     

Quantitative measurement of active polysomes of developing chick muscle

The hatching process in embryos of the toad Xenopus laevis consists of two temporally distinct phases. In phase 1, the embryo escapes sequentially from the two outermost jelly layers, J₃ and J₂, and during phase 2 the embryo hatches from the last remaining jelly coat layer J₁ and the fertilization envelope. Phase 1 hatching appears to be a physical process caused by water inbibition of jelly coat layer J₁ and dynamic changes in the volume enclosed by the fertilization envelope. The combined turgor pressure ruptures jelly coat layers J₃ and J₂. The subsequent phase 2 hatching is a result of both physical and chemical processes. Phase 1 hatching exposes layer J₁ to the medium which, in contrast to jelly layers J₂ and J₃ is partially soluble, and permits its gradual dissolution during Phase 2. The embryo secretes a proteolytic enzyme from the frontal region which partially digests the fertilization envelope; subsequent embryo movement ruptures the weakened envelope and completes the hatching process.     

Lipid dynamics in the embryos of Patiriella species (Asteroidea) with divergent modes of development

Evolution of lecithotrophic development in sea stars involved a modification in maternal provisioning from the production of yolk-dominated to lipid-dominated eggs. The dynamics of lipid reserves in the embryos of four Patiriella species differing in their lipid provisions were examined. Patiriella regularis had small yolk protein-dominated eggs (150 microm in diameter) and an ancestral mode of development through planktotrophic larvae. Patiriella calcar, Patiriella exigua and Patiriella pseudoexigua had large eggs (390-440 microm in diameter) and lecithotrophic planktonic, benthic and intragonadal larvae, respectively. Patiriella exigua deposited negatively buoyant eggs containing substantial yolk protein and lipid reserves onto the substratum. In contrast, the planktonic eggs of P. calcar and the intragonadal eggs of P. pseudoexigua were dominated by lipid and were neutrally and positively buoyant, respectively. By the blastula stage there was little trace of lipid in P. regularis embryos. Blastulae of the lecithotrophic developers, by contrast, had conspicuous lipid droplets distributed through their cells. In parallel with the change from cuboidal to columnar epithelium during the blastula to gastrula transition, lipid reserves became redistributed into the basal cytoplasm. The extent of lipid transport reflected the amount of lipid reserves. In P. pseudoexigua embryos with the greatest lipid load, basal shunting was followed by secretion of lipid into the blastocoele where it was stored for the perimetamorphic period. Evolution of lecithotrophy in Patiriella appears to reflect selection to provide metamorphic stages with nutrients normally accrued by feeding larvae with the consequence that early development is burdened by voluminous, potentially inert nutritive stores. Lipid redistribution coincident with a major developmental stage transition may be required to facilitate unimpeded morphogenesis. This phenomenon may be characteristic of lecithotrophic development in echinoderms and appears pre-adaptive for extrusion of lipid in species like P. pseudoexigua with particularly extensive lipid reserves.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Seven Hox gene sequences from the asterinid starfish Patiriella exigua (Echinodermata: Asteroidea)

Partial homeobox gene fragments were amplified from Patiriella exigua genomic DNA by PCR using degenerate primers. The primers spanned a region of 82 nucleotides within the homeobox, encompassing amino acids 21–47 of the homeodomain. The deduced amino acid sequences of the seven Hox gene sequences plus one Xlox-type gene sequence from P. exigua are presented and compared with similar sequences from other organisms. This work is a preliminary step in the analysis of the evolution of development in the Patiriella species group and the roles of Hox gene expression therein.     

Electron microscopic study of the cortical reaction of an ophiuroid echinoderm.

The egg coats of an ophiuroid echinoderm (Ophiopholis aculeata) are described by electron microscopy before and after fertilization. The unfertilized egg is closely invested by a vitelline coat about 40 A thick, and the peripheral cytoplasm is crowded with cortical granules five or six deep. During the cortical reaction, which rapidly follows insemination, exocytosis of cortical granules takes place. Some of the cortical granule material is evidently added to the vitelline coat to form a composite structure, the fertilization envelope, which is made up of a 400 A thick middle layer separating inner and outer dense layers, each about 50 A thick. The elevation of the fertilization envelope from the egg surface creates a perivitelline space in which the hyaline layer soon forms. The hyaline layer is about 2 micron thick, finely granular, and apparently derived from cortical granule material. The extracellular layers of the early developmental stages of ophiuroids and echinoids are quite similar in comparison to those of asteroids; this finding helps support Hyman's argument that the ophiuroids are more closely related to the echinoids than to the asteroids.     

The life history of Philanisus plebeius Walker (Trichoptera: Chathamiidae), a caddisfly whose eggs were found in a starfish

Abstract.

• 1 Philanisus plebeius had a univoltine life cycle with adults present from November to April in the South Island of New Zealand. Males predominated in light trap collections in all months and the mean size of adult insects declined over the flight period.
• 2 There are probably seven larval instars which inhabited the marine intertidal zone and fed primarily on non-calcareous algae. Pupae were found only on the red alga, Corallina officinalis.
• 3 Caddisfly eggs were found during most of the year in the coelom of an intertidal starfish, Patiriella regularis. Oviposition is probably through the popular pores and newly hatched larvae may leave the host via the same route or through the stomach wall.
• 4 Egg development time was greater than 30 days at 16–18°C.
• 5 P.plebeius probably colonized New Zealand from Australia after the unusual oviposition behaviour had evolved in association with the Australian cushion star, Patiriella exigua. P.regularis is less abundant and occurs lower on the shore than P.exigua and may be inaccessible to some caddisflies which oviposit amongst intertidal algae.

     

The role of the jelly coat in amino acid transport in embryos of Bombina orientalis

The obligatory role of the jelly coat for maximal transport of all amino acids, including those found to be jelly coat-independent in Xenopus laevis embryos, has been shown in Bombina orientalis embryos. Amino acid transport in dejellied embryos (without fertilization membrane and jelly coats) of Bombina, reconstituted with either intact or homogenized jelly coats, was similar to the values in normal embryos. Amino acid transport in totally dejellied embryos, and those surrounded with fertilization membrane only, was similar. Reconstitution of dejellied embryos with physically denatured jelly coats did not restore full amino acid transport. Amino acid transport values using heterologous combinations of dejellied embryos and jelly coats of Bombina orientalis and Xenopus laevis were equivalent to those in homologous combinations.     

Competition between Cellana tramoserica (Sowerby) (gastropoda) and Patiriella exigua (Lamarck) (asteroidea), and their influence on algal standing stocks

Caging experiments were undertaken in the field to test if competition occurs within and between the limpet Cellana tramoserica (Sowerby) and the starfish Patiriella exigua (Lamarck). Both species suffer from intraspecific competition, even at natural densities, and increased density reduces body weight and survival in Cellana tramoserica and growth rate in Patiriella exigua. Cellana tramoserica depresses the growth of Patiriella exigua, while the starfish unexpectedly seems beneficial to Cellana tramoserica, increasing its body weight. The difference in these effects may be due to the divergent methods of feeding used by the two species. C. tramoserica digs into the substratum and removes most available microflora, while Patiriella exigua everts its stomach onto the rock face and can only remove superficial or loosely attached microflora and detritus, and leaves a mucous web which may supplement the diet of Cellana tramoserica. Even at low densities C. tramoserica can completely prevent macroalgae from developing, while Patiriella exigua is unable to do so, although it shows the rate of development. Macroalgae grew in all the cages which contained only P. exigua, but in spite of this the starfish still suffered from intraspecific competition, probably because they cannot feed on macroalgae. Cellana tramoserica also competes by interference, and on contact with Patiriella exigua it extends its mantle and pallial tentacles, both of which may release mucus. P. exigua retreats from Cellana tramoserica, and those parts of its body that have been touched by the limpet may temporarily be immobilised and become wrinkled up. It is suggested that this interference behaviour by C. tramoserica is derived from a similar but more intense reaction that it has to invertebrate predators. Patiriella exigua lacks a pelagic larva and does not have a refuge outside the range of Cellana tramoserica. Other factors must thus be sought to explain the continued coexistence of Patiriella exigua with Cellana tramoserica. It is suggested that although C. tramoserica has a competitive effect on Patiriella exigua, it (and other grazers) may be necessary for P. exigua, preventing macroalgal growth and maintaining a surface suitable for P. exigua to feed on.     

HYBRIDIZATION OF SYMPATRIC PATIRIELLA SPECIES (ECHINODERMATA: ASTEROIDEA) IN NEW SOUTH WALES

Three species of the asteroid genus Patiriella occur sympatrically in New South Wales and the possibility for hybridization among them was examined through a series of cross-fertilization experiments. Patiriella calcar and P. gunnii are morphologically distinct as adults but indistinguishable as larvae. Patiriella exigua is morphologically distinct in both its adult and larval morphologies. The gametes of P. calcar and P. gunnii were reciprocally compatible: laboratory crosses between these species produced viable hybrid juveniles. In crosses between female P. calcar and male P. gunnii, most of the juveniles metamorphosed with an arm number intermediate between that of the parents, whereas crosses between female P. gunnii and male P. calcar produced juveniles with an arm number more similar to the maternal phenotype. Heterospecific crosses with P. exigua resulted in low fertilization rates, and viable hybrids were not produced. This species appears capable of self-fertilization. Because hybrids between P. calcar and P. gunnii were viable, neither gametic incompatibility nor hybrid inviability appears to ensure reproductive isolation between these species. Ecological or habitat segregation and temporal separation in breeding may isolate these species in the field. The results demonstrate that if gamete surface recognition molecules are involved in fertilization of P. calcar and P. gunnii, then they are not strongly species specific, at least at the sperm concentrations used in this study. Reproductive isolation between these species has evolved despite their gametic compatibility. In contrast, P. exigua is isolated from its congeners because of gametic incompatibility and several features characteristic of its reproduction and development. The implications of these findings for reproductive isolation and speciation of Patiriella and for the evolution of reproductive isolation in free-spawning marine organisms are discussed.     

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.     

Immunoelectophoretic Identification of Jelly Coat Ligands Bound by the Cortical Granule Lectin from Xenopus laevis Eggs

The formation of the fertilization layer in the Xenopus laevis egg fertilization envelope involves a lectin-ligand interaction and establishes a block to polyspermy in the extracellular matrix of the egg. The cortical granule lectin participating in the formation of the fertilization layer has been isolated but its ligand has not. We identified three jelly coat ligands bound by the cortical granule lectin using immunoelectrophoretic analyses. Two antigens were detected with anti-jelly serum and a third was identified using anti-envelope serum. All three antigenic ligands were associated with the innermost jelly coat layer, J₁, and two of the three antigenic ligands contained sulfate. One or more of these jelly coat ligands may function in establishing a block to polyspermy at fertilization in Xenopus laevis .     

Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos

Fibronectin and laminin have been found in the extracellular matrix and in the basement membrane of sea urchin embryos during early development. These glycoproteins are also found on the cell surfaces of the outer epithelial layer and on the secondary mesenchyme cells within the blastocoel. The similarity of functions of the extracellular matrix and basement membrane is discussed, as is the similarity of their molecular components. These observations suggest the possibility that fibronectin and laminin form a continuous matrix surrounding the cells which links the outer ECM (hyaline layer) to the inner ECM (basement membrane). Such a network could coordinate the various activities of the embryo during early morphogenesis.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Properties of the Cortical Granule Lectin Isolated from Xenopus Eggs

The cortical granule lectin that participates in forming the fertilization layer in Xenopus laevis was isolated and partially characterized. About 400 μg of lectin was purified from 5 mg of crude exudate by chromatography on Sepharose 6B and Concanavalin A-conjugated Sepharose 4B columns and electrophoretic separation on polyacrylamide gel. The lectin has a molecular weight of 550 Kd and is composed of two species of polypeptides (46 Kd and 42 Kd). The lectin gave a single precipitin line against material in the prefertilization layer in an agglutination reaction on an agarose plate. The agglutination reaction involved D-galactoside residues and metal ions. The lectin formed an electron-dense layer on the outer surface of the vitelline coat of oviducal eggs covered with the prefertilization layer, but on the outer surface of jelly layer, not on that of the vitelline coat of jellied eggs. Although the jelly could be agglutinated by the lectin, the possibility that the jelly layer is the site of fertilization layer formation was excluded by the fact that the prefertilization layer is the first to meet the cortical granule lectin during normal fertilization.     

Evolution of yolk protein genes in the Echinodermata

Vitellogenin genes (vtg) encode large lipid transfer proteins (LLTPs) that are typically female‐specific, functioning as precursors to major yolk proteins (MYPs). Within the phylum Echinodermata, however, the MYP of the Echinozoa (Echinoidea + Holothuroidea) is expressed by an unrelated transferrin‐like gene that has a reproductive function in both sexes. We investigated egg proteins in the Asterozoa (Asteroidea + Ophiuroidea), a sister clade to the Echinozoa, showing that eggs of the asteroid Parvulastra exigua contain a vitellogenin protein (Vtg). vtg is expressed by P. exigua, a species with large eggs and nonfeeding larvae, and by the related asterinid Patiriella regularis which has small eggs and feeding larvae. In the Asteroidea, therefore, the reproductive function of vtg is conserved despite significant life history evolution. Like the echinozoan MYP gene, asteroid vtg is expressed in both sexes and may play a role in the development of both ovaries and testes. Phylogenetic analysis indicated that a putative Vtg from the sea urchin genome, a likely pseudogene, does not clade with asteroid Vtg. We propose the following sequence as a potential pathway for the evolution of YP genes in the Echinodermata: (1) the ancestral echinoderm produced YPs derived from Vtg, (2) bisexual vtg expression subsequently evolved in the echinoderm lineage, (3) the reproductive function of vtg was assumed by a transferrin‐like gene in the ancestral echinozoan, and (4) redundant echinozoan vtg was released from stabilizing selection.