Colonial development in Pandorina morum. II. Colony morphogenesis and formation of the extracellular matrix

The colonial green alga, Pandorina morum, resembles its unicellular relative Chlamydomonas in both intracellular architecture and the composition of the extracellular matrix. Despite these similarities, cell division in Pandorina leads to the formation of a colony instead of the 8 or 16 single cells produced by cell division in Chlamydomonas. To study colony formation, partially synchronized cultures of P. morum were sampled periodically and stained with ruthenium red for electron microscopy. The cells of the colony were found to be held together during development by medial and basal connections between cells; the basal connections include strands of chloroplast. Studies of cells removed from the parental matrix before division confirmed that the cytoplasmic connections are strong enough to maintain the colonial configuration. After the medial connections break, the cells of the plate of the developing colony swing outward and attain the nearly spherical colonial configuration; the basal connections are still present. After this inversion, the formation of the extracellular matrix begins, with the colonial boundary appearing first. Capsule and sheath then form on the outer and inner faces of the colonial boundary until the extracellular matrix is complete. The process is compared to previous observations of Volvox, and possible evolutionary implications are discussed.     

Fine Structure of a Marine Ameba Associated with a Blue-Green Alga in the Sargasso Sea*,†

SYNOPSIS An ameba, bearing a fringe of scales on the plasmalemma surface, dwells among the filaments of the colonial, blue-green alga Trichodesmium thiebautii (Sournia), and preys upon bacteria growing within the colony. The cytoplasm is clearly differentiated into a fine fibrillar ectoplasm at the periphery of the cell and a central endoplasm containing most of the membranous organelles. The nucleus contains a spheroidal nucleolus which is centrally located, and a double membrane containing pores. The tubular mitochondria, microbodies, lysosomes, and endoplasmic reticulum are typical for protozoa. The Golgi apparatus consists of an array of elongate flattened cisternae. One surface is associated with a fine fibrillar layer and the opposite surface contains electron-dense vesicles (perhaps primary lysosomes) and scale-containing vesicles that appear to be the origin of the scales deposited on the plasma membrane. Three kinds of bacteria-containing vacuoles are presnt: (a) vacuoles surrounded by 3 membranes and containing bacteria that are either healthy or in an early stage of digestion, (b) singlemembrane vacuoles which are food vacuoles that become converted to digestive vacuoles, and (c) larger vacuoles resembling those in (b) which contain prey in an advanced stage of digestion. The presence of amebae within pelagic algal communities provides further evidence for the diversity of their habitats in the ocean.     

The encystment of a freshwater dinoflagellate: A light and electron-microscopical study

The process of encystment, or resting spore formation, in a freshwater dinoflagellate (Woloszynskia tylota nov. comb.) has been studied with both light and electron microscopy. The main features of the process are as follows: (i) the replacement of the theca by a thin, amorphous outer wall, which gradually thickens by the deposition of material on its inner face; (ii) the appearance of a layer of closely-packed lipid droplets at the cytoplasmic margin of the mature cyst, resembling a granular ‘inner wall’ in the light microscope; (iii) the reduction in size or disappearance of cytoplasmic structures such as chloroplasts, Golgi bodies and pusule; and (iv) the enlargement of a central ‘accumulation body’ and cytoplasmic vacuoles containing crystals. Comparisons are made with light-microscope studies of encystment of other dinoflagellates, with ultrastructural studies of non-motile division stages, with zooxanthellae and with fossil dinoflagellate cysts or hystrichospheres.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : I. Studies on Cultured Macrophages Exposed to Chloroquine

Mouse macrophages exposed to 30 µg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1–4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.     

Colonial development in Pandorina morum. I. Structure and composition of the extracellular matrix

Every cell in a colony of the freshwater alga Pandorina morum produces a daughter colony in each round of vegetative reproduction. The cells of P. morum structurally resemble those of Chlamydomonas and do not obviously differ from their presumptive unicellular ancestor in ways that would account for their colonial state. It is therefore possible that their colonial association may be the result of altered extracellular matrix, intercellular connections, or modified processes of cell division and matrix formation. The ultrastructural studies and chemical analysis reported here show that the matrix of P. morum is a multilayered structure containing hydroxyproline-rich glycoproteins and sulfated polysaccharide. Significantly, this matrix displays no major differences from the “wall” of Chlamydomonas species. No intercellular connections have been found in mature colonies. The extracellular matrix therefore maintains, but does not initiate, the colonial arrangement of the cells.     

GLOEOCOCCUS TETIMSPORUS SP. NOV. (TETRASPORALES,PALM ELL ACEAE), FROM COLORADO MOUNTAIN LAKES1

A new species, Gloeococcus tetrasporus sp. nov., collected from mountain lakes, is described from unialgal culture. Vegetative cells are ellipsoid and Chlamydomo nas–like, occur in tetrad complexes within the general colonial matrix, and exhibit slow, limited motility within the confines of the individual gelatinous matrices. The colonial matrix is amorphous and structureless, without a definite bounding layer. Colonies may reach several centimeters in size. Vegetative cells have a parietal cup–shaped chloroplast with a central–basal pyrenoid and a small, linear stigma, two contractile vacuoles, and two short flagella. Cell division is by eleutheroschisis in nonflagellate cells. After two divisions, four daughter cells arc formed within the expanded parent wall that will become incorporated into the colonial matrix. Zoospores are formed either from transformed vegetative cells or after cytokinesis. Zoospore flagella are two to three times the length of vegetative cell flagella. Rapid flagellar movement ruptures the sheath and liberates the zoospores. When zoospores settle, they secrete new sheaths, and divide twice to initiate new colonies. Sexual reproduction and formation of resistant spores were not observed.     

Cleavage, incomplete inversion, and cytoplasmic bridges in Gonium pectorale (Volvocales, Chlorophyta)

Multicellularity arose several times in evolution of eukaryotes. The volvocine algae have full range of colonial organization from unicellular to colonies, and thus these algae are well-known models for examining the evolution and mechanisms of multicellularity. Gonium pectorale is a multicellular species of Volvocales and is thought to be one of the first small colonial organisms among the volvocine algae. In these algae, a cytoplasmic bridge is one of the key traits that arose during the evolution of multicellularity. Here, we observed the inversion process and the cytoplasmic bridges in G. pectorale using time-lapse, fluorescence, and electron microscopy. The cytoplasmic bridges were located in the middle region of the cell in 2-, 4-, 8-, and 16-celled stages and in inversion stages. However, there were no cytoplasmic bridges in the mature adult stage. Cytoplasmic bridges and cortical microtubules in G. pectorale suggest that a mechanism of kinesin-microtubule machinery similar to that in other volvocine algae is responsible for inversion in this species.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

The fine structure of myelinated nerve cell bodies in the bulbus olfactorius of man

Summary In the bulbus olfactorius of man numerous myelinated nerve cell bodies occur in the stratum plexiforme internum and stratum granulosum internum. In many respects they resemble the neighbouring granule cells: small chromatin clumps border on more than half of the circumference of the nucleus, the thin cytoplasmic rim contains abundant polysomes and sometimes pigment complexes with numerous light vacuoles, the cells often show a process which extends up to the stratum glomerulosum, the perikarya are devoid of synaptic contacts whereas the proximal segment of the peripheral processes display rare contacts. The myelin sheath varies in thickness, consisting of 2 to 24 lamellae with distances between the major dense lines ranging from 9.3 to 11.3 nm. The myelin sheath may enclose the cell body completely or partially and accompany the proximal segment of the process arising from the perikaryon. On partially enveloped perikarya, the myelin lamellae end in formations like those of the node of Ranvier, though often less regularly. Within the compact myelin sheath all of its lamellae may be distended for a short distance by glial cytoplasm as in the Schmidt-Lanterman incisures of peripheral nerve fibres. Adjacent to the outermost myelin lamella myelinated axons and cell bodies, tentatively identified as oligodendrocytes, as well as granule cells may be closely joined.Supported by the Deutsche Forschungsgemeinschaft (Br. 634/1)     

Ultrastructure of Naegleria fowleri enflagellation.

Amoebae of Naegleria fowleri nN68 became elongated flagellated cells 150 to 180 min after subculture to non-nutrient buffer. N. fowleri NF69 did not become elongated or flagellated under these conditions. Electron microscopic examination of N. fowleri confirmed that it is a typical eucaryotic protist with a distinct nuclear envelope and prominent nucleolus, numerous vacuoles and cytoplasmic inclusions, pleomorphic mitochondria, and some rough endoplasmic reticulum. During incubation in non-nutrient buffer, both strains lost ultraviolet-absorbing material to the medium, and the number of vacuoles decreased. In strain nN68, basal bodies, a rootlet, and flagella are formed quickly after an initial lag of 90 min. Initially, the rootlet is not associated with the nucleus but they become associated subsequent at the leading end of the elongated cell. In elongated cells, the rootlet lies in a furrow or groove extending the length of the nucleus. Flagella of N. fowleri nN68 exhibit the typical 9 + 2 arrangement of filaments and are surrounded by a sheath which is continuous with the plasma membrane. The enflagellation process in N. fowleri can be manipulated reproducibly.     

Regulation and control of intracellular algae (= zooxanthellae) in hard corals

To examine algal (= zooxanthellae) regulation and control, and the factors determining algal densities in hard corals, the zooxanthellae mitotic index and release rates were regularly determined in branch tips from a colony of a staghorn coral, Acropora formosa, recovering from a coral ''bleaching'' event (the stress-related dissociation of the coral–algal symbiosis). Mathematical models based upon density-dependent decreases in the algal division frequency and increases in algal release rates during the post-bleaching recovery period accurately predict the observed recovery period (ca. 20 weeks). The models suggest that (i) the colony recovered its algal population from the division of the remaining zooxanthellae, and (ii) the continual loss of zooxanthellae significantly slowed the recovery of the coral. Possible reasons for the ''paradoxical'' loss of healthy zooxanthellae from the bleached coral are discussed in terms of endodermal processes occurring in the recovering coral and the redistribution of newly formed zooxanthellae to aposymbiotic host cells. At a steady-state algal density of 2.1 x 10⁶ zooxanthellae cm^-2 at the end of the recovery period, the zooxanthellae would have to form a double layer of cells in the coral tissues, consistent with microscopic observations. Neighbouring colonies of A. formosa with inherently higher algal densities possess proportionately smaller zooxanthellae. Results suggest that space availability and the size of the algal symbionts determines the algal densities in the coral colonies. The large increases in the algal densities reported in corals exposed to elevated nutrient concentrations (i.e between a two- and five-fold increase in the algal standing stock) are not consistent with this theory. We suggest that increases of this magnitude are a product of the experimental conditions: reasons for this statement are discussed. We propose that the stability of the coral–algal symbiosis under non-stress conditions, and the constancy of zooxanthellae densities in corals reported across growth form, depth and geographic range, are related to space availability limiting algal densities. However, at these densities, zooxanthellae have attributes consistent with nutrient limitation.     

Prey Capture and Phagocytosis in the Choanoflagellate Salpingoeca rosetta

Choanoflagellates are unicellular and colonial aquatic microeukaryotes that capture bacteria using an apical flagellum surrounded by a feeding collar composed of actin-filled microvilli. Flow produced by the apical flagellum drives prey bacteria to the feeding collar for phagocytosis. We report here on the cell biology of prey capture in rosette-shaped colonies and unicellular “thecate” or substrate attached cells from the choanoflagellate S. rosetta. In thecate cells and rosette colonies, phagocytosis initially involves fusion of multiple microvilli, followed by remodeling of the collar membrane to engulf the prey, and transport of engulfed bacteria into the cell. Although both thecate cells and rosette colony cells produce ∼70 nm “collar links” that connect and potentially stabilize adjacent microvilli, only thecate cells were observed to produce a lamellipod-like “collar skirt” that encircles the base of the collar. This study offers insight into the process of prey ingestion by S. rosetta, and provides a context within which to consider potential ecological differences between solitary cells and colonies in choanoflagellates.     

Video plankton recorder reveals high abundances of colonial Radiolaria in surface waters of the central North Pacific

Colonial spumellarian Radiolaria are heterotrophic protiststhat form large (up to several meters in length), gelatinousstructures in the surface waters of all tropical and subtropicaloceanic ecosystems. These species are morphologically and trophicallycomplex and some, but not all, produce silica skeletal structuresof considerable paleontological significance. Skeletonless speciesof Radiolaria are poorly sampled by plankton nets, which canseverely damage these delicate organisms. Therefore, abundancesof colonial Radiolaria typically have been underestimated inquantitative studies of zooplankton abundance and biomass. Herewe document the abundances of colonial Radiolaria in the centralNorth Pacific based on analysis of video images from a miniaturizedvideo plankton recorder. We observed abundances of radiolariancells in colonies that exceeded previous reports of total Radiolariaby more than ten-fold, and counts of skeleton-bearing Radiolariaby more than two to three orders of magnitude. Biomass (carbon)within these colonies was similar to or greater than the totalradiolarian biomass (i.e. including all solitary species) previouslyreported for the Pacific. Symbiont productivity within colonialRadiolaria was estimated to constitute a modest but significantfraction of total primary productivity (up to     

Ultrastructural and histochemical investigation of the terminal capillaries in the spleen of the carp (Cyprinus carpio L.)

Summary In the spleen of the carp arterial capillaries of a highly differentiated structure have been studied by light and electron microscopy. These capillaries share various structural characteristics with the sheathed capillaries (ellipsoids of Schweigger-Seidel) of higher vertebrates. The long arterial capillaries of the carp spleen are provided with cuboidal endothelial cells containing filaments approximately 7 nm in diameter. There is no basal lamina. The endothelial cells form various types of cell junctions, but there are also extensive areas without any junctions. Here, a free passage is possible between the capillary lumen and the subendothelial space. The capillaries possess a single-layered sheath of macrophages. Characteristically, the sheath macrophages possess long and slender cell processes forming a loose framework, the meshes of which are filled with lymphocytes and spindle cells. The sheath macrophages show a zone of ectoplasm rich in filaments. They also contain numerous phagolysosomes rich in hydrolytic enzymes, as identified histochemically. The sheath is sharply limited against the pulp by a thick layer of collagen fibers.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

ULTRASTRUCTURAL OBSERVATIONS ON THE TRICHOBLASTS OF EQUISETUM

Equisetum trichoblasts are densely cytoplasmic, containing numerous starch-containing plastids, mitochondria, and concentrations of rough endoplasmic reticulum with attached polysomes. Numerous vesicles of Golgi origin are present, containing a lightly staining fibrillar material; these vesicles appear to fuse with the wall. The outer tangential and radial walls become thickened while the inner tangential wall remains thin with numerous plasmodesmata. As the trichoblasts develop into root hairs, vacuolation occurs, resulting in large vacuoles. This may represent autolytic vacuolation. The cytoplasm of the root hairs is similar to that of the trichoblasts.     

Two distinct steps for spontaneous generation of subprotoplasts from a disintegrated bryopsis cell

The unusual nature of protoplasm to generate subprotoplasts spontaneously from disintegrated Bryopsis cells was examined. Protoplasm extruded from algal cells aggregated rapidly in cell sap which was derived mainly from huge central vacuoles of the cells. Electron microscopic observations revealed extensive agglutination of algal cellular membranes in the protoplasmic masses, suggesting that this is of primary importance for the wound-healing ability of the alga. Seawater caused spheration of the resultant protoplasmic aggregates. Gelatinous sheaths were formed temporarily surrounding the spherical protoplasmic masses before reformation of cell membrane. Staining with phosphotungstic and chromic acids suggested that new cell membrane was formed by fusion of the disintegrated original cell membrane with cytoplasmic vesicles on the surfaces of the protoplasmic masses. Both pH and salts were found to be essentially important at the two steps of subprotoplast generation. The newly formed cell membranes were responsible for subsequent notable plasmolysis of the wounded cells in seawater. Thus, it is suggested that unicellular marine algae Bryopsis spp. naturally contain effective materials for agglutinating and fusing particular cellular membranes through the sequential aid of acidic cell sap and alkaline seawater after disintegration of the giant cells.     

Schistosoma mansoni: penetration apparatus and epidermis of the miracidium

Free swimming miracidia of Schistosoma mansoni were studied with the electron microscope for the purpose of describing the penetration apparatus and the epidermis. The penetration apparatus was composed of 3 unicellular glands which contain membrane-bound vesicles containing macromolecular diglycols. Each gland contained a nucleus, rough endoplasmic reticulum, Golgi complexes, numerous mitochondria, and glycogen stores. Each gland cell opened to the exterior through the apical papilla.The surface of the miracidium, with the exception of the apical papilla, was covered with ciliated epidermal cells containing numerous mitochondria, membranous bodies, and glycogen. No nuclei were detected within these epidermal cells. Intercellular ridges connecting with subepidermal cytons interrupted the epidermal cells at numerous points. The ridges were joined to the epidermal cells by septate desmosomes. Beneath the epidermal cells were found circular and longitudinal muscle bundles.Sensory structures of various types were associated with the outer covering. These consisted of (1) numerous “knob-like” cytoplasmic projections associated with epidermal cells, (2) bulbous, lamelloid structures with external cytoplasmic projections, and (3) ciliated nerve endings with posterior epidermal tiers and ciliated nerve pits associated with apical papilla.