Egg capsules of a parasitic turbellarian flatworm: ultrastructure of hatching sutures.

Egg capsules of Syndisyrinx franciscanus, an intestinal parasite of sea urchins (Strongylocentrotus spp.), consist of a bulb, which contains the embryos, and a stalk-like filament. The wall of the bulb is about 12 microns thick and is composed of sclerotized proteins. The end of the bulb opposite the attachment of the filament bears a reticulum of hatching sutures. Transmission electron microscopy discloses that hatching sutures traverse the entire thickness of the capsule wall. The inner 9-10 microns of sutures are a uniform 20 nm in width and contain a trilaminar cementum. The outer 2-3 microns of sutures are 15 nm to more than 500 nm in width and contain an electron-lucent cementum. The latter may contain an irregular, median, electron-dense layer or, more commonly, electron-dense granules. The outside of some capsules is partially covered by a thin, electron-dense material. A previous study showed that sutures in intact capsules of Syndisyrinx franciscanus are not affected by host digestive fluids, but are severely weakened immediately prior to hatching owing to activities of the embryos. The hypothesis that the embryos secrete a hatching enzyme is supported by findings that sutures of intact capsules are not affected by externally applied trypsin, but become weakened when capsules are cut open and then incubated in trypsin. Scanning electron microscopy reveals that the outer parts of sutures often remain intact after hatching. We hypothesize that the ability of sutures to resist enzymatic attack from the outside, but not the inside, results from differences in the chemical properties of the cementums in outer and inner parts of sutures.

The influence of tunichrome and other reducing compounds on tunic and fin formation in embryonic Ascidia callosa Stimpson.

Tunic in 46-hr-old Ascidia callosa larvae reared from dechorionated neurulae is either markedly reduced in thickness or absent altogether. The epidermis is fragile and cuticular fins fail to develop. Dechorionated neurulae treated with tunichrome and other reducing compounds (e.g., glutathione, ascorbate) show an enhancement in tunic formation and rudimentary fin development. UV absorbance spectra of extracts from unfertilized eggs, late tail-bud embryos, and tadpole larvae indicate that tunichrome may be present in all developmental stages. Experiments with neurulae in which the chorion was punctured with tungsten needles but not removed signify that the test cells are the most likely source of tunichrome. Results are consistent with the hypothesis that tunichrome is involved in the natural processes of tunic morphogenesis in ascidian embryos.

OVULATION AND THE FINE STRUCTURE OF THE STICHOPUS CALIFORNICUS (ECHINODERMATA: HOLOTHUROIDEA) FECUND OVARIAN TUBULES.

The ovary of Stichopus californicus consists of several size classes of tubules, which insert into a central gonad basis. The largest tubules contain the oocytes that will be spawned in the current season. All tubules are composed of three layers. Outermost is a complex peritoneum composed of epithelial cells, axons and muscle cells. The fine structure of the peritoneal neurons suggests their involvement in neurosecretory activity. Between the basal laminae of the peritoneum and the inner epithelium is the ovarian connective tissue compartment, including the genital hemal sinus. This sinus probably conveys nutrients from the periphery of the tubule to oocytes located deep within. The inner epithelium is composed of parietal and follicular epithelial cells and the oocytes. Stichopus oocytes contain three classes of microtubules based upon their location, orientation, and lability during fixation. Microtubules from the apical protuberance encircle the germinal vesicle. Cortical microtubules lie just under the cell surface and run parallel to it. Deep cytoplasmic microtubules run radially from the interior of the oocyte towards the cell surface. Oocytes are held within follicles by junctional complexes until the time of ovulation. Ovulation can be monitored in severed follicles of this species because an oolamina insures follicle integrity after detachment from the ovary. The onset of ovulation is marked by the dissolution of junctional complexes. This is followed by a cytochalasin B sensitive contraction of the follicle cells. The follicle contracts down around the oocyte, to lie collapsed against the ovarian wall while the oocyte is free within the ovarian lumen.

Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility.

Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistent direction with an average rate of 22.5 micron/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless , and the rate decreased to an average of 14.5 micron/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations . After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damaged centrioles. The results presented in this paper suggest that the centrosome is an important structure in controlling the rate and direction of newt eosinophil motility.

Chiton integument: ultrastructure of the sensory hairs of Mopalia muscosa (Mollusca: Polyplacophora).

The dorsal integument of the girdle of the chiton Mopalia muscosa is covered by a chitinous cuticle about 0.1 mm in thickness. Within the cuticle are fusiform spicules composed of a central mass of pigment granules surrounded by a layer of calcium carbonate crystals. Tapered, curved chitinous hairs with a groove on the mesial surface pass through the cuticle and protrude above the surface. The spicules are produced by specialized groups of epidermal cells called spiniferous papillae and the hairs are produced by trichogenous papillae. Processes of pigment cells containing green granules are scattered among the cells of each type of papilla and among the common epidermal cells. The wall or cortex of each hair is composed of two layers. The cortex surrounds a central medulla that contains matrix material of low density and from 1 to 20 axial bundles of dendrites. The number of bundles within the medulla varies with the size of the hair. Each bundle contains from 1 to 25 dendrites ensheathed by processes of supporting cells. The dendrites and supporting sheath arise from epidermal cells of the central part of the papilla. At the base of each trichogenous papilla are several nerves that pass into the dermis. Two questions remain unresolved. The function of the hairs is unknown, and we have not determined whether the sensory cells are primary sensory neurons or secondary sensory cells.

Ascidian larval tunic: Extraembryonic structures influence morphogenesis.

The larval tunic of Corella inflata is composed of two cuticular layers, extracellular filaments and ground substance. It lies outside the epidermis and most of it is known to be produced by the epidermis. The dorsal, ventral and caudal fins are specialized parts of the tunic that are essential for larval locomotion. The following hypothesis was tested: Morphogenesis of the larval fins is dependent upon the presence of extraembryonic structures (test cells, chorion or follicle cells) before completion of the late tail bud stage of development. We tested this by dechorionating embryos of Corella inflata and Ascidia paratropa. The operation removes all extraembryonic structures. It was performed mainly on neurula, early tail-bud and late tail-bud stages. Fin formation is inhibited when neurulae are dechorionated but not when late tail-bud or older embryonic stages are dechorionated. Dechorionated neurulae produce all of the major components of the tunic (cuticular layers, filaments and ground substance) but they are unable to form functional fins. At the time of dechorionation, in all experiments, the embryos had no fins. Removal of the follicle cells does not inhibit fin formation. The test cells are known to secrete granular "ornaments" that attach to the surface of the tunic. The fibrous, acellular chorion may serve to contain the test cells and their products or products of the embryo that are not firmly attached. The test cells may induce or control the morphogenesis of the larval fins in ascidians before the late tail-bud stage of development. We suggest ways of testing this hypothesis and an alternative hypothesis.

Rhythmic contractions of the ampullar epidermis during metamorphosis of the ascidian Molgula occidentalis.

The ampullae of Molgula occidentalis are hollow, tubular extensions of the epidermis. They are ensheathed by a secreted tunic. When they grow out shortly after settlement, the ampullae spread the tunic over the substratum to form a firm attachment for the sessile juvenile. A simple squamous epithelium forms the thin ampullar walls. A glandular, simple columnar epithelium forms the distal tip of each ampulla. The glandular cells probably secrete the adhesive that attaches the tunic to the substratum. Repetitive, peristaltic contractions pass from the base to the distal end of each ampulla. Microsurgery, time-lapse cinemicrography and TEM have been used to analyze this phenomenon. The contractions are mediated by a layer of 4-8 nm microfilaments in the base of the ampullar epithelium. Each juvenile has 7-9 ampullae which contract at different frequencies. Isolated ampullae continue to contract normally for several days. Thus each ampulla has an intrinsic rhythm. Microsurgical experiments suggest that there is no specific region within an ampulla with unique pacemaker properties. It is proposed that communication via gap junctions allows the coordination of ampullar cells into a well organized peristaltic wave.

Reflector cells in the skin of Octopus dofleini.

The cells that form the reflecting layer beneath the chromatophore organs of the octopus are conspicuous elements of its dermal chromatic system. Each flattened, ellipsoidal reflector cell in this layer bears thousands of peripherally radiating, discoidal, reflecting lamellae. Each lamella consists of a proteinaceous reflecting platelet enveloped by the plasmalemma. The lamellae average 90 nm in thickness and have variable diameters with a maximum of about 1.7 micrometer. Sets of reflecting lamellae are organized into functional units called reflectosomes. The lamellae in each reflectosome form a parallel array - similar to a stack of coins. The average number of lamellae in a reflectosome is 11. Adjacent lamellae are uniformly separated by an extracellular gap of about 60 nm in embedded specimens. The reflectosomes are randomly disposed over the surface of the reflector cell. The observed organization of the reflectosomes is compatible with its role as a quarter-wave thin-film interference device. The alternating reflecting lamellae and intelamellar spaces constitute layers of high and low refractive indices. Using measurements of the thicknesses and refractive indices of the platelets and interlamellar spaces, we have calculated that the color of reflected light should be blue - green, as seen in vivo. The sequence of events leading to the definitive arrangement of the reflectosomes is uncertain. The reflector cells of O. dofleini are compared and contrasted with the iridophores of squid.

The spermatozoa of ascidians: acrosome and nuclear envelope.

The spermatozoon of Ascidia callosa has a head with a wedge-shaped tip. Between the nuclear envelope and the plasmalemma, at the tip of the head, there are one or two previously undescribed vesicles, 45 to 55 nm in diameter. These vesicles have the characteristics of an acrosome. Their role in the process of fertilization has not been determined. Ultrastructural studies of sperm activation are needed, but claims that the spermatozoa of ascidians do not have an acrosome should be reconsidered. Behind the tip of the sperm there are pores in the nuclear envelope. This part of the envelope also contains a dense band of amorphous material that may have a supportive function. A nearly identical structure, associated with pores has been found in the spermatozoon of Boltenia villosa. An analysis of the nuclear envelope of Ascidia callosa indicates that the same structure has previously been misinterpreted as an acrosome in the spermatozoon of Ascidia nigra.

Larval adhesive organs and metamorphosis in ascidians. II. The mechanism of eversion of the papillae of Distaplia occidentalis.

The cup-shaped adhesive papillae of Distaplia occidentalis evert at the onset of metamorphosis and each transforms into a hyperboloidal configuration. The rate of transformation is a function of temperature. At 14 degrees C complete eversion takes about 30 seconds. Myoepithelial cells that extend from the rim to the base on the cup contract. Simultaneously the central part of the papilla advances 60--70 micrometers. During the last phases of eversion, collocytes (cells that secrete adhesives) on the inner wall of the cup and on the sides of the axial protrusion flow outward and form a collar-like structure. The myoepithelial cells contain arrays of thick and thin filaments. These become compacted during contraction. The surfaces of these cells become extensively folded as they shorten to about 1/3 of rest length. According to the proposed model the myoepithelial cells are the driving force in papillary eversion. Immediately after eversion is completed the papillae begin to retract. Eversion of the papillae is not inhibited by cytochalasin B, but the process of retraction is reversibly inhibited. Some histological characteristics of five types of everting papillae in four families of ascidians are compared.

Larval adhesive organs and metamorphosis in ascidians. I. Fine structure of the everting papillae of Distaplia occidentalis.

The larava of Distaplia occidentalis bears three cup-shaped adhesive papillae, each with a prominenta axial protrusion, At the onset of metamorphosis these organs rapidly evert through fenestrations in the cuticular layers of tunic exposing hyaline caps of adhesive. Additional adhesive material is secreted from collocytes during eversion. The stickiness of the papillae facilitates attachment to a variety of substrates. Each papilla is composed of more than 900 cells; six different types were identified. The wall of the cup contains about 260 myoepithelial cells with long attenuated processes. These extend from the rim of the cup to the base in the parietal (inner) layer. The apices of the myoepithelial cells are held in place by 11 pairs of specialized anchor cells bearing long bulbous microvilli. When the myoepithelial cells contract they force the axial protrusion forward and transform the papilla into a hyperboloidal configuration. The papilla is innervated by small motor fibers, but sensory fibers were not detected. The adhesive papillae of Distaplia are discussed in relationship to nine other recognizable types of papillae in the ascidians.

Ultrastructure and differentiation of ascidian muscle. I. Caudal musculature of the larva of Diplosoma macdonaldi.

The larval caudal musculature of the compound ascidian Diplosoma macdonaldi consists of two longitudinal bands of somatic striated muscle. Approximately 800 mononucleate cells, lying in rows between the epidermis and the notochord, constitute each muscle band. Unlike the caudal muscle cells of most other ascidian larvae, the myofibrils and apposed sarcoplasmic reticulum occupy both the cortical and the medullary sarcoplasm. The cross-striated myofibrils converge near the tapered ends of the caudal muscle cell and integrate into a field of myofilaments. The field originates and terminates at intermediate junctions at the transverse cellular boundaries. Close junctions and longitudinal and transverse segments of nonjunctional sarcolemmata flank the intermediate junctions, creating a transverse myomuscular (TMM) complex which superficially resembles the intercalated disk of the vertebrate heart. A perforated sheet of sarcoplasmic reticulum (SR) invests each myofibril. The sheet of SR spans between sarcomeres and is locally undifferentiated in relation to the cross-striations. Two to four saccular cisternae of SR near each sarcomeric Z-line establish interior (dyadic) couplings with an axial analogue of the vertebrate transverse tubular system. The axial tubules are invaginations of the sarcolemma within and adjacent to the intermediate junctions of the TMM complex. The caudal muscle cells of larval ascidians and the somatic striated muscle fibers of lower vertebrates bear similar relationships to the skeletal organs and share similar locomotor functions. At the cellular level, however, the larval ascidian caudal musculature more closely resembles the vertebrate myocardium.