Scanning and transmission electron microscopic studies of jejunal microvilli of the rat, hamster and dog

Scanning and transmission electron microscopy were used to investigate the surface area and number per unit area of microvilli from jejunal villus epithelial cells in the rat, hamster and dog. The calculated mean microvillus surface area was 0.419 μ², 0.573 μ², 0.751 μ² for the rat, hamster and dog respectively. The largest number of microvilli per square micron freeze dried villus surface was measured in the rat with a mean value of 65. Hamster and dog freeze dried specimens had lower mean values of 54 and 34 microvilli respectively. The total microvilli surface area in square micron per square micron villus surface was more closely related for the three species with values of 27.23 for the rat, 30.94 for the hamster and 25.53 for the dog. These data indicate an inverse relationship between the mean microvillus surface area and population density in the species studied. However, the total microvilli surface area per unit villus surface is relatively similar for the three species. The observed number of microvilli per unit villus surface was shown to vary depending upon the dehydration technique employed for preparation of scanning electron microscopic specimens. This variation probably reflects shrinkage artifact and should be considered in soft tissue studies involving the scanning electron microscope.     

Physiological response of Spisula subtruncata (da Costa, 1778) to different seston quantity and quality

Individuals of the bivalve Spisula subtruncata were fed a mixed diet comprising of sea water enriched with the diatom Phaeodactylum tricornutum and ashed silt within a range of concentrations, simulating natural conditions above pseudofaeces threshold. The designed ranges for total particulate matter were between 10 and 30 mg l^–1and organic content of seston 15–40%. Filtration rate, rejection rate, ingestion rate and absorption rate were measured at those different conditions. Filtration rate and rejection rate were significantly correlated to total particulate matter and percentage of organic matter, with higher rates at higher values of total particulate matter and lower values of percentage organic matter. Ingestion rate was maintained at similar levels in all the treatments and organic enrichment of the ingested food occurred due to preingestive selection of the filtered material. A differential absorption rate occurred at different levels of organic matter in the diet with high rates at high values of the organic content of the diet. S. subtruncata showed different physiological responses to changes of the food conditions: (1) Increase of pseudofaeces production at increasing levels of particulate matter, (2) preingestive selection of organic material which enriched the organic fraction of ingested food, (3) stabilized ingestion rate and (4) increase of the absorption rate at high organic levels of the seston.     

Structure and formation of the adventitious tube of the Japanese watering-pot shell Stirpulina ramosa (Bivalvia, Anomalodesmata, Clavagellidae) and a comparison with that of the Penicillidae

Abstract. Stirpulina ramosa is the only extant endobenthic representative of the Clavagellidae and is restricted to the waters of Japan. A single intact adventitious tube of this species has been obtained and its structure is described. The right valve is 16 mm long and located within the adventitious tube. It has an opisthodetic ligament located on resilifers. There are anterior and posterior adductor muscle scars, a thick pallial line, and pallial and pedal gape (right valve only) sinuses. The left shell valve is but 9 mm long and is united into the fabric of the adventitious tube via the intermediary of a shelly saddle. Internally, only the anterior adductor muscle scar and a small element of the pallial line scar are identifiable on the left valve. The posterior adductor and the rest of the pallial line scar (including a pallial sinus) are, remarkably, located on the adventitious tube beyond the shell valve margin. The adventitious tube of S. ramosa is formed in a manner wholly dissimilar from that of Brechites vaginiferus (Penicillidae). In B. vaginiferus, the tube is secreted as a single entity from the general outer mantle surface, including the siphons, covering the body. As a consequence, both shell valves are incorporated into the structure of the tube and the watering pot is bilaterally symmetrical. In S. ramosa, the tube and watering pot are secreted from the mantle margin and surface surrounding and extending from the left shell valve, so that only the left valve is incorporated into its structure. A dorsally derived mantle element is progressively extended over to the right side of the body, meeting a ventrally derived counterpart that passes beneath it, forming a pleat in the calcareous structure of the right side of the tube that they secrete. This pleat extends into the complex of watering‐pot tubules and forms the pedal gape. The watering pot is thus Ω shaped. The ventrally derived mantle element forms a sinusoidal crest on the right‐hand base of the watering pot, creating a pedal gape sinus scar on the right valve. The Clavagellidae radiated widely in the Mesozoic, leaving behind a rich fossil record for Stirpulina. Only S. ramosa, however, has survived until the present. In contrast, the Cenozoic Penicillidae has a poor fossil record, but there is a rich variety of extant endobenthic watering‐pot shells. It has been argued hitherto that the two families represent a remarkable example of convergent evolution. In view of the success of the Penicillidae and thus the endobenthic, tube‐dwelling lifestyle, however, it is hard to understand why Stirpulina has largely died out—even S. ramosa being known by but one or two specimens. A study of the anatomy of S. ramosa might one day answer this question.     

Anatomy of Afropomus balanoideus (Mollusca,Gastropoda, Ampullariidae) and its implications for phylogeny and ecology

Summary The hitherto unknown anatomy of Afropomus balanoideus is described. Its systematic position within the Ampullariidae is indicated by the possession of the following autapomorphic characters of the Ampullariidae: presence of a lung sac, an ampulla-shaped aorta anterior, a bipartite copulatory organ of pallial origin, a left-sided zygoneur nervous system, a bipartite kidney, a lamellate osphradium, labial tentacles, an ingestion and an egestion sipho and a radula with strong teeth. The pallial vas deferens is completely closed, while the pallial oviduct is anatomically open in its distal part but functionally closed, because it is embedded in the surrounding mantle tissue. The copulatory organ is of pallial origin and innervated by the right pleural ganglion. The plesiomorphic states of some characters indicate that Afropomus balanoideus might represent the adelphotaxon of the remaining Ampullariidae. The morphological structures show that this species is adapted to an amphibious mode of life and to aestivation, as has been shown for other species of Ampullariidae.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lobster,Homarus americanus. An electron microscope-protein tracer study

The present investigation describes the ultrastructural changes which occur at the surface and in the cytoplasm of developing oocytes of the lobster, Homarus americanus, during vitellogenesis. The immature oocytes showed no surface specializations of the oolemma and no pinocytotic activity was observed. Horseradish peroxidase (HRP) tracer studies showed penetration of the tracer into the perivitelline space, but no uptake by the oocytes. The surfaces of oocytes examined during vitellogenesis, when yolk protein accumulation was maximal, exhibited numerous microvilli that projected into the perivitelline space, often appearing to be embedded in the follicular cell mass. In addition, the plasma membrane of vitellogenic oocytes contained many pinocytotic pits frequently situated at the bases of microvilli. The perivitelline space was engorged with electrondense material which appeared similar to that contained in pinocytotic structures of the oocytes. Vitellogenic oocytes incubated in HRP showed uptake of tracer reaction product by the coated pits and vesicles of the oolemma. Aggregation and subsequent fusion of these vesicles into large multivesicular bodies of ingested material were also observed in vitellogenic oocytes. Animals artificially induced to undergo vitellogenesis exhibited modulations of oocyte ultrastructure similar to those of normal vitellogenesis, notably, pinocytotic incorporation of extra-oocytic material and hypertrophy of oocyte surface microvilli. This study supports the hypothesis for a dual source of yolk protein in the American lobster.     

An Ultrastructural and Cytochemical Study of the Cell Surface of a Suctorian Ciliate1

The surfaces of the main cell body, tentacle shaft, and knob of Discophrya collini, a freshwater suctorian ciliate, were characterized using various cytochemical techniques. Cells prepared for conventional transmission electron microscopy exhibited a 50–60 nm thick fuzzy layer over the cell body surface; this layer was absent from the tentacle knob. A thick (240 nm), two-layered surface coat surrounding the main cell body was stained with ruthenium red. This heavy coat was absent from the surface of the knob where a thin, dense, ruthenium red-positive layer and projecting filaments were present. Freeze-etched material revealed a “particle region” (150–250 nm in thickness) closely associated with the outer cell surface of the suctorian. Fixed specimens were treated with four different lectins and analyzed with electron microscopy in order to obtain information about the carbohydrate composition of the outer surface of D. collini. Concanavalin A bound to the surface of the cell body and tentacle shaft as a dense, particulate layer (80 nm thick) but thinned to 13–16 nm over the surface of the knob. Wheat germ agglutinin-treated cells also displayed a heavy, electron-dense layer (128 nm thick) that surrounded the main cell body and tentacle shaft, but only scattered patches of bound wheat germ agglutinin were observed on the surface of the knob. Discophrya treated with Helix agglutinin or peanut agglutinin appeared similar to control cells. Suctorians were treated with lectins in vivo in an attempt to inhibit capture and ingestion of their prey, Tetrahymena pyriformis, by masking prey receptor sites on the knob. Concanavalin A and, to a lesser degree, wheat germ agglutinin, successfully inhibited attachment of the prey organism. Helix agglutinin and peanut agglutinin had little effect on prey capture.     

东方田鼠与昆明小鼠乳腺癌细胞表面特征比较

目的　通过对封闭群东方田鼠 (Microtusfortis)和昆明小鼠乳腺癌细胞扫描电镜特征的观察 ,旨在了解两者之间的差异。方法　将乳腺癌肿块与经产正常东方田鼠乳腺以及昆明小鼠自发性乳腺癌肿块作常规扫描电镜。结果　正常东方田鼠乳腺的腺管结构规则 ,可见分散的乳腺细胞 ,乳腺细胞表面有许多皱褶和不规则的小丘状突起 ,状如桑椹 ,无明显可见的微绒毛 ;东方田鼠乳腺癌的腺管结构不规则 ,细胞表面密布微绒毛 ,微绒毛长而不规则 ,末端成松叶状 ;昆明小鼠乳腺癌的组织中散布有大量带有微绒毛的癌细胞 ,其微绒毛特点为密而短 ,末端为圆纯状。结论　东方田鼠与昆明小鼠乳腺癌细胞表面呈现不同的微绒毛特征     

Influence of diet on pre-ingestive particle processing in bivalves: II. Residence time in the pallial cavity and handling time on the labial palps

Previous studies have demonstrated that bivalve molluscs respond to changing food conditions through processes such as preferential selection and ingestion of particulate matter. Little is known, however, about the underlying mechanisms accountable for these responses. To further explain feeding processes at the level of the pallial organs, we determined pallial cavity residence times, or the amount of time it took particles to travel from the inhalant aperture to the stomach, in two species of bivalves, Crassostrea virginica and Mytilus edulis, under conditions of differing particle quality, particle concentration, and temperature. From these residence times, particle-handling times on the labial palps were determined. Diets of three different qualities were tested, including Rhodomonas lens cells, particles prepared from ground Spartina sp. detritus, and a 50/50 mixture of both. Bivalves were delivered one of the three diets along with 10-μm fluorescent polystyrene beads (tracer), removed from feeding chambers at intervals from 30 s up to 20 min, and placed in liquid nitrogen to halt particle transport. Digestive systems of bivalves were then dissected and examined for the presence of tracer beads. Particle-residence times in the pallial cavity and handling times on the labial palps of C. virginica were significantly affected by changes in diet type. Particle-handling times on the palps decreased with increasing diet quality and ranged from 2.2 min (100% R. lens) to 22.8 min (100% ground Spartina sp.), accounting for 88% and 99%, respectively, of the total time particles spent in the pallial cavity. In contrast, diet quality had little effect on particle-residence times in the pallial cavity of M. edulis. However, residence times were affected by temperature and diet concentration. Temperature significantly affected residence times at particle concentrations of both 20 and 100 particles μl⁻¹, whereas particle concentration affected residence times at 20 °C, but not at 5 °C. Particle-handling times on the labial palps ranged from less than 1 to 5.5 min, depending on temperature and concentration, accounting for 50% to 82%, respectively, of the total time particles spent in the pallial cavity. We suggest that (1) observed interspecific differences in particle handling on the labial palps may be due to differences in palp morphology and function, and (2) particle sorting and selection on the labial palps is a rate-limiting step of pre-ingestive feeding processes in by bivalves.     

Comparative study on the surface ultrastructures of fertilized eggs of three Sebastes species

Fertilized eggs of Sebastes schlegelii, Sebastes pachycephalus and Sebastes hubbsi are morphologically similar under stereomicroscope. However, under the scanning electron microscope, significant differences in the ultrastructures of the egg surface among the three species were observed, and herein, a taxonomic key was proposed for future applications. A clustering analysis based on the ultrastructures of the egg surface and the diameter of the oil globule suggested that among the three species, S. hubbsi and S. pachycephalus were more genetically similar, while S. schlegelii had diverged earlier. The results agree with the conclusions drawn from morphological and molecular analyses on adult samples of the same species.     

Morphological studies on the mantle of the fresh-water mussel Amblema (UNIONIDAE): Scanning electron microscopy

The scanning electron microscope has been used to describe the surface morphology of the mantle in mantle-shell preparations from the fresh-water mussel Amblema. In some regions (adductor muscle insertions), the mantle is firmly attached to the shell. In other areas (along the main course of the mantle), transient adhesions between the outer mantle epithelial cells and the nacre appear to temporally further compartmentalize the extrapallial fluid possibly as a prerequisite for the initial crystallization phenomenon. At the mantle edge, as well as at the isthmus, the periostracum was seen to extrude from the periostracal groove. At the siphonal edge, peculiar fingerlike processes were identified; these may represent primitive photoreceptors. The epithelial cells of the outer mantle epithelium are all microvillated whereas those of the inner mantle epithelium are both microvillated and ciliated. Specific differences in surface morphology are described for various regions of the outer mantle epithelium. These may be related to precise regionalized functional differences of this tissue. Several functions of the mantle, in addition to shell formation, and based on its various morphologies, are also discussed.     

Scanning electron microscopy of the rat retina

Summary Fixed and unfixed, freeze-dried pieces of isolated retina and the posterior part of the eye bulb from adult rats were examined in a scanning electron microscope.The inner limiting membrane shows distinct cell borders, protrusions, and scattered microvilli-like structures. Different types of nerve cells are observed in the ganglion cell layer and the inner nuclear layer. They all lack synaptic boutons on the surface of their perikarya. There is an intercellular space between the processes in the nerve fiber layer.The inner and outer segments are surrounded by a space with extracellular material. Their surface is smooth or slightly undulated. There is no evidence indicating the existence of basal infoldings continuous with the membraneous structures inside the rod outer segments. The connecting piece between the inner and outer segments resemble a symmetrically shaped hour-glass. The surface of the epithelial cells is covered by microvilli forming a honeycomb-like structure and each outer segment is surrounded by several microvilli.The results obtained are discussed in relation to those obtained by transmission electron microscopy. The probable existence of a significant extracellular space and the distribution of extracellular material between the segments and the microvilli are discussed.Supported by grants from H. Jeanssons Stiftelse Riksföreningen mot Cancer (265-B69-OIX) and the Swedish Medical Research Council (B70-12X-2534-02). I would like to thank the Swedish Silicate Research Institute, Göteborg, for using their scanning electron microscope, and Miss. M. Persson for skilful technical assistance.     

Ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans (Müller) (mollusca,prosobranchia) and the ommatophore of Helix pomatia L. (Mollusca,Pulmonata)

The ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans and Helix pomatia is very similar. Complex apical structures are present, and the lateral plasma membrane exhibits three zones: (1) a zone of slight interdigitations; (2) a zone characterized by longitudinal plicae; (3) a zone of basal radiculae. The portions of the sensory cells located within the epithelial layer are accommodated in longitudinal grooves in the supporting cells. However, there are also differences. In Pomatias elegans the apical surface is differentiated into long microvilli that are sometimes dichotomously branched and invested by a surface coat along their entire length. Cytofilia and cilia of the sensory cells pass through this layer of microvilli and surface coat throughout its entire width. In Helix pomatia the supporting cells are somewhat smaller and the apical differentiation consists of candelabra-like protrusions, which are usually three times dichotomously branched. The final branchings, corresponding to microvilli, are called terminal twigs. They are covered by a surface coat, which forms a feltwork. The cytofilia and cilia of the sensory cells that intertwine among the protrusions are confined to the space below the terminal twigs, where they compose the spongy layer.     

Direct innervation of the mantle edge gland by neurosecretory axons in Helisoma duryi (Mollusca: Pulmonata)

Summary The mantle edge gland of Helisoma duryi is innervated by neurosecretory axons from the pallial nerves. Synaptoid contacts occur between axons and gland cells, and there is ultrastructural evidence for the release of neurosecretory material. The mantle edge gland contributes to the deposition of periostracum during shell formation, and direct neurosecretory innervation may control shell growth and regeneration.Supported by a National Research Council of Canada Grant (A-4673) and Negotiated Grant D-61     

Nutritive phagocytosis in animal cells

Summary Nutritive phagocytosis in the hydroid Clava squamata was studied with the electron microscope, using carbon particles of 0.6 as an indicator.An early step in phagocytosis is the transformation, in many cells, of the free border from a type with cylindrical microvilli to one with a complicated system of cytoplasmic folds.Particles fixed at the actual stage of ingestion are found (a) between two cytoplasmic folds, (b) between a fold and a relatively straight portion of the cell surface, or (c) in a depression of an otherwise straight portion of the cell surface.Ingested carbon particles were always found enclosed by a membrane, with a layer of moderate electron density between the carbon particle and the membrane.The ingested carbon particles are localized apically in small vesicles each containing one particle (interpreted as primary phagocytic vesicles) or at deeper levels of the cell, in larger vesicles containing many carbon particles (interpreted as secondary phagocytic vesicles).Other cytoplasmic changes during phagocytosis relate to the distribution of mitochondria and the occurence and distribution of flattened vesicles of a characteristic appearance.With the technical assistance of Birgitta af Burén.Financial support from Swedish Natural Science Research Foundation is gratefully acknowledged.     

Some aspects of the fine structure of the statocysts of the molluscsPecten andPterotrachea

Summary The statocyst ofPecten is composed of hair cells and supporting cells. The hair cells bear kinocilia and microvilli at their distal ends and the supporting cells bear microvilli. The cilia have a 9+2 internal filament content, and arise from basal bodies that have roots, basal feet and microtubular connections. Two different ciliary arrangements are described, one with a small number of cilia arranged in a ring, and another with many more cilia arranged in rows. Below the hair cells are probable synapses. A ciliated duct connects to the lumen of the static sac and passes through the centre of the static nerve. The hair cells in the statocyst ofPterotrachea bear kinocilia and microvilli. The possible importance of cilia and microvilli in the transduction process is discussed.We would like to thank ProfessorJ. Z. Young for bringing specimens ofPterotrachea from Naples and also the staff of the Stazione Zoologica for the provision of specimens, Dr.M. Land for providing specimens ofPecten, the Science Research Council (U.K.) for providing the electron microscope used in much of the study and also for a grant to one of us (V.C.B.), and Mrs.J. Parkers and Mr.R. Moss and Mrs.J. Hamilton for much photographic and technical assistance.     

Sorting out of sea urchin embryonic cells according to cell type

Experiments carried out on a mixture of blastomeres from two different species of echinoderms, Arbacia punctulata and Echinarachnius parma, demonstrate that reaggregation is species-specific and that sorting out of blastomeres according to species occurs. Transmission electron microscope analysis of the reaggregation of suspensions of blastomeres from each species separately and together in mixed suspension reveals that cell adhesion in these forms displays a species-specific morphology. Arbacia blastomeres make initial contact and adhere to one another by means of microvilli and the formation of an intercellular hyaline-like material characteristic of the species. Echinarachnius blastomeres form a scalloped edge at the surface of two apposing blastomeres and form a specific intercellular hyaline-like material. Blastomeres of the two different species adhere to one another but form neither microvilli nor a scalloped border. No hyaline-like material is formed between blastomeres of the two different species.     

The biology and functional morphology of Arca noae (Bivalvia: Arcidae) from the Adriatic Sea, Croatia, with a discussion on the evolution of the bivalve mantle margin

In the Croatian Adriatic, Arca noae occurs from the low intertidal to a depth of 60 m; it can live for > 15 years and is either solitary or forms byssally attached clumps with Modiolus barbatus. The shell is anteriorly foreshortened and posteriorly elongate. The major inhalant flow is from the posterior although a remnant anterior stream is retained. There are no anterior but huge posterior byssal retractor muscles and both anterior and posterior pedal retractors. The ctenidia are of Type B(1a) and the ctenidial–labial palp junction is Category 3. The ctenidia collect, filter and undertake the primary sorting of potential food in the inhalant water. The labial palps are small with simple re‐sorting tracks on the ridges of their inner surfaces. The ciliary currents of the mantle cavity appear largely concerned with the rejection of particulate material. The mantle margin comprises an outer and an (either) inner or middle fold. The outer fold is divided into outer and inner components that secrete the shell and are photo‐sensory, respectively. The latter bears a large number of pallial eyes, especially posteriorly. The inner/middle mantle fold of A. noae, possibly representative of simpler, more primitive conditions, may have differentiated into distinct folds in other recent representatives of the Bivalvia.     

In vitro culture of glochidia from the freshwater mussel Anodonta cygnea

Abstract. Larvae of the freshwater swan mussel, Anodonta cygnea , were cultured in artificial media at the controlled temperature of 23°±2°C, with successful metamorphosis for the first time. The artificial medium contained a mixture of M199, common carp plasma, and antibiotics/antimycotics. Glochidia were reared to the juvenile stage in the medium after 10–11 d of culture. After 15 d of controlled feeding with phytoplankton, the juveniles showed an elongated shell with several growth lines. Larval survival was 34.3±9.3%, whereas the proportion undergoing metamorphosis was ≤60.8±4.2%. The ultrastructure of early developmental stages was observed by scanning electron microscopy, from the glochidial to the juvenile stage. Glochidia had a hooked shell, with two equal triangular valves formed by a calcareous layer with numerous pores and covered by a thin cuticle of chitin–keratin. The appearance of the complete foot within 11 d of in vitro culture was considered the final feature of metamorphosis to the juvenile stage. The main alteration during juvenile development was the formation, under the glochidial shell, of a new periostracum with growth lines. The prominent foot, gradually covered by long, dense cilia, showed rhythmical movements involved in the capture of particulate matter. Similarly, cilia and microvilli present in the mantle also performed the same role. Longer cilia, sparsely distributed in the mantle, may function as chemotactile sensors.