A Metamorphic Inducer in the Opisthobranch Haminaea callidegenita: Partial Purification and Biological Activity.

Larvae of Haminaea callidegenita (Mollusca: Cephalaspidea) were induced to metamorphose by a compound found in the gelatinous matrix composing most of the egg mass. A functionally similar compound isolated from adult tissue also induced metamorphosis in H. callidegenita larvae. Opisthobranchs are frequently induced to metamorphose by a specific prey item or a substrate characteristic of the adult habitat, but this is the first known instance of metamorphosis occurring in response to a compound produced by adult conspecifics. The inducer was purified from egg mass jelly (EMJ) by high pressure liquid chromatography (HPLC) and was found to be smaller than 1000 Da, polar, non-proteinaceous, and very stable. We isolated a compound of identical activity from egg masses produced by four other opisthobranch species, suggesting that the same or chemically similar compounds are intrinsic to opisthobranch egg masses. However, only H. callidegenita larvae metamorphosed in response to EMJ. Competent larvae of five other mollusc species did not respond to the partially purified EMJ inducer but did respond to a specific substrate associated with each species. The presence of the inducer within the egg mass causes an unusual developmental pattern in H. callidegenita, a poecilogonous species that produces both swimming veliger and crawling juvenile offspring.

Isolation of Neurons of a Nudibranch Veliger.

A technique was developed to dissociate and culture identified sensory neurons and interneurons from the anterolateral propodial ganglia of metamorphically competent veligers of the nudibranch, Onchidoris bilamellata. Receptor cells have been represented as receiving an environmental cue that initiates the settlement response. The ganglionic cells, along with other cell-types from the propodial region housing the ganglia, were excised with a large-bore micropipette, and dissociated by mild trypsin incubation and trituration. Cells and tissues were plated in poly-L-lysine-coated plastic culture dishes containing modified Aplysia medium and survived for up to four days. The different cell-types possess diagnostic features, so they can be recognized under culture conditions. Sensory cells were bipolar in profile, with one end of the cell being thickened, representing the receptor apparatus. Interneurons are unipolar or bipolar in shape, and bear thin neurites. Other cell-types, including myocytes, ciliated epidermal cells, nonciliated epidermal cells, and gland cells were identified. Identifications of living cells were corroborated through electron microscopical analysis.

Kinetics of Fertilization in the Sea Urchin Strongylocentrotus franciscanus: Interaction of Gamete Dilution, Age, and Contact Time.

Determining fertilization success of free spawning organisms in the field requires knowledge of how eggs and sperm interact under varying encounter frequencies and durations. In the laboratory, we investigated the relative influence of sperm concentration, egg concentration, sperm-egg contact time, and sperm age on fertilization in the sea urchin Strongylocentrotus franciscanus. Our results indicated that sperm concentration, sperm-egg contact time, sperm age, and individual variability were sequentially the most important factors influencing fertilization success. Egg concentration was not significant over the range tested. A theoretical model of fertilization (Vogel-Czihak-Chang-Wolf model) was used to estimate the two rate constants of fertilization kinetics: the rate constant of sperm-egg encounter and rate constant of fertilization. This model explained 91% of the variation in fertilization success, provided estimates of the rate constants involved in fertilization, and indicated the proportion (3%) of sperm-egg contacts that result in fertilization. Estimates of sperm swimming velocity and egg diameter were used to independently calculate the rate of sperm-egg encounter and confirm the predictions of the model. This model also predicts the non-significant effect of egg concentration on fertilization success found empirically.

Fertilization in a chiton: acrosome-mediated sperm-egg fusion.

Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome-mediated sperm-egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (= chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.

Fine structure of Sertoli cells in three marine snails with a discussion on the functional morphology of Sertoli cells in general.

The fine structure of Sertoli cells in three marine prosobranch molluscs has been studied with light- and electron microscopy. Sertoli cells of prosobranchs are modified columnar epithelial cells that maintain continuous contact with the basal lamina and extend from it to the lumen of a testicular tubule. Spermatogenesis takes place between adjacent Sertoli cells, but a continuous layer of cytoplasm separates the spermatogonia from the basal lamina, thus restricting the basal compartment to spermatogonium mother cells. Substances traversing the basal lamina from the interstitial space must pass either through or between the Sertoli cells. However, between the cells, a permeability barrier composed of septate and desmosome-like junctions blocks the passage of substances, such as the tracer lanthanum nitrate. The basally-located nucleus is irregularly shaped with fine granular euchromatin and some peripheral heterochromatin: satellite karyosomes border the nucleolus. There is an extensive intracellular digestive system that is used effectively to phagocytize waste sperm and residual cytoplasm. Cytoplasmic processes of Sertoli cells penetrate throughout the germinal epithelium. In some prosobranchs that exhibit sperm polymorphism these processes must coordinate to bring together a clone of eupyrene sperm and a carrier sperm at a particular time in development. The only cytoskeletal elements available within the processes to generate such movements are microtubules. We propose that the term 'nurse cell', which has been used in the past to describe at least three different cell types, including Sertoli cells and apyrene sperm, be restricted to abortive oogonia that contribute to development of an oocyte.

A technique for obtaining replicas of small tubular systems by microinjection of vinyl acetate.

A microtechnique has been devised for replicating tubular systems of small organisms. In this case, the reproductive system of a small marine snail was replicated by inserting a micropipette through the gonopore and filling the genital ducts with vinyl acetate from a flow-controlled syringe. Subsequently the tissue was dissolved and the model was photographed. The technique could be adapted to a variety of different systems, particularly small invertebrates such as insects, annelids and molluscs.

Fine structure of the larval rhinophores of the nudibranch, Rostanga pulchra, with emphasis on the sensory receptor cells.

The rhinophores of the veliger larva of Rostanga pulchra are located in the intravelar field near the base of the velar lobes. Each rhinophore is a cylindrical structure, tapering distally, and covered with a dense meshwork of microvilli. A conspicuous row of ciliary tufts runs along each side of the rhinophore and several stiffer tufts, composed of fewer cilia, are positioned around the tip or at the base. The rhinophoral epithelium consists of supporting cells, ciliated cells (giving rise to the ciliary rows), dendritic terminals (giving rise to the tufts around the apex), and sinuses containing occasional amebocytes. The lumen of the rhinophore is occupied by the rhinophoral ganglion and muscle cells that are oriented in two perpendicular planes. Cells bodies of the dendritic endings are located within the rhinophoral ganglion, which in turn joins into the optic and cerebral ganglia. Rhinophoral ganglionic neurons do not synapse with each other, but numerous neuromuscular synapses are found in the lumen of the rhinophore. Morphological evidence suggests that the dendritic endings are chemoreceptors and the ciliated cells are possibly mechanoreceptors but are not functional at this stage in development. The functional role of the rhinophores is discussed in relation to larval behavior at settlement and metamorphosis.

The fine structure of the polymorphic spermatozoa of Fusitriton oregonensis (Mollusca: Gastropoda), with notes on the cytochemistry of the internal secretions.

The prosobranch Fusitriton oregonensis exhibits an unusual form of sperm polymorphism. The viable, eupyrene sperm are attached in groups of about fifty to worm-shaped, apyrene, carrier sperm. There is a second apyrene sperm, which is lancet-shaped and has a different internal organization than the carrier, but does not transport eupyrene sperm. The eupyrene sperm are filiform (185 microns long), with a conical acrosome, elongate nucleus and midpiece. They contain large stores of glycogen in the principal piece, together with an unusually high proportion of protein. The latter is due to a complex interconnecting system of fibres that supports the tail internally. A distinct annulus is located, characteristically, at the junction between midpiece and principal piece. The carrier sperm has a core of about 112 axonemes that arise from basal bodies in the anterior end and extend through its entire length of 36 microns. The basal bodies have unstriated rootlets that are embedded in a granular cap. Large membrane-bound "yolk bodies" are arranged along the length of the carrier sperm, on either side of the median axonemal core. Dense bodies, which may be indigestible residues formed from the degeneration of the nucleus, are excreted by exocytosis. Individual carrier sperm are capable of "corkscrew" propulsion, resembling that of spirochaetes. The lancet sperm is three times as long as the carrier. The sixteen or so axonemes, which are arranged peripherally like a cage enclosing the cytoplasm, originate from a dense centriolar plate in the anterior end. The cytoplasm is filled with secretions including small yolk granules, dense bodies (also excreted), clear vesicles, and a membranated granular secretion that resembles mucus. The possible functions of the lancet and carrier sperm are discussed.

Locomotion of the filiform sperm of littorina (Gastropoda, Prosobranchia).

The filiform sperm of Littorina sitkana swims effectively in sea water and more viscous fluids, overcoming the problems of a non-uniform flagellar beat with an unusual mechanism, which involves three main events: (1) the sperm rotates anti-clockwise (looking from tail to head); then (2) stops rotating and stiffens itself to form a screw-shape, with the tail being held perpendicular to the middle piece, and finally; (3) reverses its rotation and propels itself forward in a clockwise spiral. The average velocity of sperm is approximately 184 micrometer/s with a rotational frequency of 24 revs/s. The mechanism of propulsion may involve two kinetic centers at opposite ends of the sperm, which coordinate their movements to produce anti-clockwise rotation, stationary twisting, or clockwise rotation, in a manner reminiscent of spirochaetes. Littorina sperm also exhibit slower methods of propulsion including swimming backwards (tail first) at 18 micrometer/s, and "gliding" at about 30 micrometer/s. The adaptive significance of the rapid propulsion is not obvious, because Littorina copulate and fertilize internally and at each stage in the transfer there are external aids to sperm transport, such as ciliary action (oviduct) and muscular expulsion (bursa and seminal receptacle). The filiform shape, however, is well-adapted for long-term storage in the female body. These points are discussed.

Fine structural study of the statocysts in the veliger larva of the nudibranch, Rostanga pulchra.

The two statocysts of the veliger larva of Rostanga pulchra are positioned within the base of the foot. They are spherical, fluid-filled capsules that contain a large, calcareous statolith and several smaller concretions. The epithelium of the statocyst is composed of 10 ciliated sensory cells (hair cells) and 11 accessory cells. The latter group stains darkly and includes 2 microvillous cells, 7 supporting cells, and 2 glial cells. The hair cells stain lightly and each gives rise to an axon; two types can be distinguished. The first type, in which a minimum of 3 cilia are randomly positioned on the apical cell membrane, is restricted to the upper portion of the statocyst. The second type, in which 9 to 11 cilia are arranged in a slightly curved row, is found exclusively around the base of the statocyst. Each statocyst is connected dorso-laterally to the ipsilateral cerebral ganglion by a short static nerve, formed by axons arising from the hair cells. Ganglionic neurons synapse with these axons as the static nerve enters the cerebral ganglion. The lumen of the statocyst is continuous with a blind, constricted canal located beneath the static nerve. A diagram showing the structure of the statocyst and its association with the nervous system is presented. Possible functions of the statocyst in relation to larval behavior are discussed.

Morphogenesis of larval cuticle in the polychaete Phragmatopoma lapidosa. A correlated scanning and transmission electron microscopic study from egg envelope formation to larval metamorphosis.

The development of the egg envelope and its incorporation into the larval cuticle of the polychaete Phragmatopoma lapidosa, was studied by correlative scanning and transmission electron microscopy. The mature egg possesses an envelope composed of five zones including an outer granular zone formed by the tips of the egg microvilli. The formation of the granules is described and their functions are discussed. The entire egg envelope is retained as the larval cuticle up to the 16 h trochophore stage. From this stage to about the 60 h larval stage, the envelope is gradually lost and replaced by a cuticle consisting of branching microvilli. The cuticle of the 20 day larva is composed of highly branching microvilli penetrating a homogeneous electron opaque cuticle. The possible functions of the cuticle among the Annelida are discussed.

Coelomic pouch formation in the starfish Pisaster ochraceus (Echinodermata: Asteroidea).

The process of coelomic pouch formation in Pisaster ochraceus was studied with light microscopy, transmission and scanning electron microscopy and time-lapse cinemicrography as well as with the drug cytochalasin B. As in most asteroids, the paired coelomic pouches of Pisaster ochraceus are formed from outpocketing of the archenteron. Arrays of 50 Å microfilaments are found in the presumptive coelomic pouch cells at the apex of the archenteron as well as in the filopodia of the mesenchyme cells. Both cell types undergo active movements throughout the entire process. Treatment of embryos with cytochalasin B (CCB) during coelomic pouch formation results in the loss of cell movements and the regression of the coelomic pouches; this is accompanied by the loss of microfilament arrays in both cell types. Cell movements and microfilament arrays reappear on removal of CCB and coelomic pouch formation resumes. Our evidence suggests that the microfilaments in the presumptive coelomic pouch cells provide the main force for the outpocketing movement. The major role of the microfilament arrays in the filopodia of the mesenchyme cells associated with the coelomic pouches is to determine the definitive shape and location of the pouches.

On the nurse cell and the spermatozeugma in Littorina sitkana.

Nurse cells develop from diploid cells in the testis. Each cell undergoes a reduction division which leaves the nucleus with half the volume of a normal diploid cell. They send out pseudopodia which form desmosome-like junctions with developing spermatids. The nurse cells detach from the testicular wall, their nuclei degenerate and secretion droplets form in the cytoplasm. The pseudopodia are drawn in as the cytoplasmic secretions swell and the nurse cell becomes spherical. The eupyrene sperm become grouped unilaterally and at this stage are attached to the nurse cell by only the tips of their acrosomes. At maturity the nurse cells with their clumps of attached eupyrene sperm (spermatozeugmata) are released from the testis via ducts into the seminal vesicles,where they are stored prior to copulation. Nurse cells serve similar functions to those of apyrene sperm which are common among the Molluscs. We believe that the nurse cell and apyrene sperm are homologous.

Fine structural observations of sperm resorption in the seminal vesicle of a marine snail, Littorina scutulata (Gould, 1849).

Waste sperm and spermatozeugmata in the seminal vesicles of Littorina scutulata are phagocytised either by cell buds (large vesicles given off from the epithelial cells) or by the epithelial cells themselves. Cell buds containing sperm, are in turn engulfed by epithelial cells. In both cases, heterophagic vacuoles are formed inside of the cell and subsequently the vacuoles are fused with primary lysosomes or lysosomal derivatives to become secondary lysosomes. Throughout this process the sperm are being digested. The second lysosome transforms further to telolysosome and finally to residual body when the sperm is completely digested.

Spermatogenesis of a marine snail, Littorina sitkana.

The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.

Sand dollar: a weight belt for the juvenile.

Juvenile sand dollars (Dendraster excentricus) selectively ingest heavy sand grains from the substrate and store them in an intestinal diverticulum which may function as a weight belt, assisting the young animal to remain in the shifting sandy environment. The sand disappears from the diverticulum when the animal reaches the length of 30 millimeters.