The use of fluorochromes in the cytochemical characterization of some phytoflagellates

Summary Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (incuding the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies inA. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca²⁺ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca²⁺ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates ofP. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.     

THE ULTRASTRUCTURE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The freshwater dinoflagellate Glenodiniopsis steinii Wolsoszyńska was examined using computer-assisted three-dimensional reconstruction of serially sectioned cells observed with the transmission electron microscope and images from the scanning electron microscope. Vegetative cells contain ultrastructure typical of freshwater dinoflagellates including trichocysts, mitochondria, Golgi bodies, starch grains, and lipid bodies. The chloroplast is a single, multilobed structure, not multiple discoid chloroplasts as previously described. The “C” shape of the nucleus is apparently due in part to the size and location of the pusule.     

SEM survey on mediterranean species of Podolampas

Abstract

In this work we have considered the features of thecal plates of some mediterranean species of Podolampas. The thecal plates have pores different in size and distribution according to their position and to the species considered. We have found some constant structures: kind of perforations of the apical, preequatorial and postequatorial thecal plates. The antiapical thecal plates have both constant and distinctive specific features.     

Development of compound trichocysts in the ciliatePseudomicrothorax dubius

Summary During the logarithmic growth of the ciliatePseudomicrothorax dubius associations between mitochondria, rough endoplasmic reticulum and dictyosomes have been observed. The Golgi apparatus is very active and it is suggested that, as a consequence of cytotic activity, the contents of the Golgi vesicles become incorporated into large irregular vacuoles as globular material. The large vacuoles develop into trichocysts and the dictyosome derived globules consolidate to ultimately form the rod-like arms of the trichocysts of theMicrothoracidae.     

Quantitative analysis of ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.)

Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.     

ROOT APEX STRUCTURE IN EPHEDRA MONOSPERMA AND EPHEDRA CHILENSIS (EPHEDRACEAE)

The root meristem of E. monosperma and E. chilensis possesses a central group of distinctive, large cells. These cells have large nuclei with scattered heterochromatin, proplastids with no starch, small vacuoles, mitochondria, few dictyosomes and endoplasmic reticulum cisternae, and lipid deposits. Over a 24 hr labelling period, the large cells fail to incorporate ³H-thymidine, whereas cells both distal and proximal to this region do. A quiescent center which includes these large cells is present therefore. Both species have an extensive root cap, the length being contributed by mitoses in many tiers of cells distal to the quiescent center. The root cap consists of a columella and peripheral regions. Distinctive amyloplasts, an increase in the number of endoplasmic reticulum cisternae and dictyosomes, large vacuoles, and lipid deposits are characteristic of differentiated columella cells. Peripheral cells elongate, lose most of their starch, and are eventually sloughed from the root.     

ULTRASTRUCTURE OF THE DINOFLAGELLATE PERIDINIUM CINCTUM F. OVOPLANUM. II. LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS ON FERTILIZATION

Fertilization in Peridinium cinctum f. ovoplamtm has been investigated at both the light and electron microscopic levels. Gamete formation occurs when vegetative cells are placed into nitrogen deficient media. The majority of gametes observed possess thin thecal plates; however, some are naked. Gametes have few chloroplasts as compared to vegetative cells, numerous membrane bounded storage bodies, many starch grains, and chromosomes which appear slightly unwound. Gamete fusion is observed to peak 7–10 days after inoculation into nitrogen deficient media. Fusion occurs in an area of the sulcus devoid of reticulate thecal plates at or adjacent to the flagellar pores. A fertilization tube is formed and proceeds to widen along the sulcus. Karyogamy occurs within the fertilization tube before plasmogamy is completed. The resulting planozygote is a two walled structure containing two longitudinal flagella. It enlarges over a 2-week period giving rise to the hypnozygote.     

Physiological and ultra-structural changes in Brassica napus seedlings induced by cadmium stress

The effects of cadmium on physiological and ultrastructural characteristics were evaluated in 6-d-old seedlings of two Brassica napus L. cultivars Zheda 619 and ZS 758. Results show that Cd at lower concentration (100 μM) stimulated the seedling growth but at higher concentration (500 μM) inhibited the growth of both cultivars, decreased content of photosynthetic pigments, activities of antioxidant enzymes, and increased the content of malondialdehyde and reactive oxygen species. Cd content in different parts of seedlings was higher in ZS 758 than in Zheda 619. Electron micrographs illustrated that 500 μM Cd severely damaged the leaf and root tip cells of both cultivars. Under Cd stress, the size and number of starch grains, plastoglobuli, and lipid bodies in the chloroplasts increased. In the root tip cells, enlarged vacuoles, diffused cell walls, and undeveloped mitochondria were detected.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF A NEW MARINE SAND‐DWELLING PROROCENTRUM SPECIES,P. TSAWWASSENENSE (DINOPHYCEAE,PROROCENTRALES), FROM BRITISH COLUMBIA,CANADA1

A new marine benthic, sand‐dwelling Prorocentrum species from the temperate region of the Pacific coast of British Columbia, Canada, is described using LM and EM and molecular phylogenetic analyses. The cells have a broad oval shape, 40.0–55.0 μm long and 30.0–47.5 μm wide, and a wide U‐shaped periflagellar area on the right thecal plate. The left thecal plate consists of a straighter apical outline in the form of a raised ridge. Five to six delicate apical spines in the center of the periflagellar area are present. The nucleus is located in the posterior region of the cell, and a conspicuous pusule is located in the anterior region of the cell. The cells have golden‐brown chloroplasts with a compound, intrachloroplast pyrenoid that lacks a starch sheath. The thecal plates are smooth with round pores of two different sizes. The larger pores are arranged in a specific pattern of radial rows that are evenly spaced around the plate periphery and of irregular rows (or double rows) that form an incomplete “V” at the apical end of the plates. Large pores are absent in the center of the left and right thecal plates. The intercalary band is striated transversely and also has faint horizontal striations. Trichocysts and two types of mucocysts are present. The molecular phylogenetic position of Prorocentrum tsawwassenense sp. nov. was inferred using SSU rDNA sequences. This new species branched with high support in a Prorocentrum clade containing both benthic and planktonic species.     

Ultrastructure of the testicular gland of Blennius pavo (Pisces,Teleostei)

Summary The testicular gland of Blennius pavo Risso, 1810 is an accessory sex organ situated ventrally, adjacent to the testis. Sperm have to cross it to reach the vas deferens. Changes in the fine structure of the gland were monitored monthly over a period of 1 year. The annual cycle is closely correlated to the reproductive cycle. Maximum synthetic activity and cell size are reached during late prespawning and early spawning. At that time the number of lipid vacuoles increases markedly. Vesicles of smooth endoplasmic reticulum ER, numerous Golgi cisternae and tubulovesicular mitochondria which are characteristics of steroid producing cells can be observed. Secretory vesicles are synthesized in the apical region of the cells and then released into the ducts of the gland. The content of lipids is maintained until the resting period. The supranuclear region of the cells contains accumulations of lamellar bodies that probably derive from lysosomal vacuoles. Fine structural characteristics suggesting the function of the testicular gland are discussed.     

Ultrastructure and development of nonarticulated laticifers in seedlings ofEuphorbia maculata L.

The ultrastructure of nonarticulated laticifers in the seedlings ofEuphorbia maculata was studied at various developmental stages. The apical regions of the seedling laticifers growing intrusively contained large nuclei with mainly euchromatin and dense cytoplasm possessing various and many organelles such as rich ribosomes, several small vacuoles, giant mitochondria with dense matrices, rough endoplasmic reticulum, dictyosomes, and proplastids. This result suggested that the apical regions of laticifers were metabolically very active. Laticifers in seedlings at the first-leaf developmental stage did not contain latex particle. In seedlings at second-leaf growth stage, the laticifer cells contained numerous and elongated small vacuoles. These vacuoles appeared to arise by dilation of the endoplasmic reticulum and frequently possessed osmiophilic or electron-dense latex particles. The small vacuoles fused with the large vacuole occupying the central portion of the subapical region of laticifers, and then the latex particles were released into the large central vacuole. The latex particles varied in size and were lightly or darkly stained. Proplastids with a dense matrix and a few osmiophilic plastoglobuli were filled with an elongated starch grain and thus were transformed into amyloplasts. Latex particles were initially produced in the laticifers after seedlings had developed their second young leaves. In seedlings at forth-leaf stage, latex particles with an alveolated rim were found in the laticifers.     

POTENTIAL TOLERANCE MECHANISMS OF PROROCENTRUM MICANS (DINOPHYCEAE) TO SUBLETHAL LEVELS OF COPPER1

We investigated how Prorocentrum micans Ehrenberg, a planktonic dinoflagellate common in Portuguese coastal waters, is able to tolerate and recover from sublethal concentrations of copper(II). The experimental design simulated events in inshore waters, where P. micans is subjected to high levels of pollutants, including copper. Decrease in growth rate, induction of a growth lag phase, temporary loss of motility, and potassium leakage were the effects induced in P. micans cultures by 90 nM labile copper. A 10–20-fold increase in cellular copper concentration was observed in toxicity experiments. Copper efflux (representing a 50% decrease in cellular metal content) was a short-term tolerance mechanism. A 25-kDa protein was detected after only 3 h of exposure to copper, but there was no evidence of phytochelatin synthesis. Ultracytochemical labeling of metals with the sulfide-silver procedure showed that copper was associated with the thecal plates, starch grains, and, to a lesser extent, lipid droplets. High values affixation capacities and average conditional stability constants for copper binding by starch, amylopectin, and cellulose support the location of copper in thecal plates and starch grains. We conclude that P. micans responds rapidly to copper toxicity and has two tolerance mechanisms for copper: copper efflux and sequestration in polymeric substances.     

Fine Structure of the Fat Body and its Bacteroids in Blattella germanica (Blattodea)

The abdominal fat body of the cockroach Blattella germanica contains three characteristic cell types—trophocytes, bacteriocytes and urate cells—which have been investigated by electron microscopy. The trophocytes are rich in lipid droplets of different sizes; glycogen, rough endoplasmic reticulum and mitochondria are also abundant. In females immediately after eclosion, the trophocytes contain a greater number of lipid droplets, some of which have different electron density; glycogen and cytoplasmic organelles are clearly reduced. The bacteriocytes hold rod-like and spherical bacteroids, which are encapsulated by a vacuolar membrane; they show a thin cytoplasmic membrane and an evident cell wall surrounded by a membrane-like outer envelope. The bacteroids appear to be dividing either by transverse partition or by budding. The urate cells, adjacent to the bacteriocytes, are characterized by complex urate vacuoles delimited by a double layer-structure.     

ULTRASTRUCTURAL OBSERVATIONS OF GYRODINIUM ESTUARIALE (DINOPHYCEAE)1

Gyrodinium estuariale Hulburt has ultrastructural features typical for a dinoflagellate including a particularly well developed pusule and numerous prominent trichocysts. The amphiesma is relatively simple with thecal vesicles containing thecal membranes. The epicone is embellished with two concentric raised ridges reminiscent of the form typical for more heavily thecate genera.     

The fine structure of the liver cells in the bat (Myotis myotis) during hibernation,arousal and forced feeding

Summary The ultrastructure of liver cells was studied in three groups of bats (Myotis myotis) captured in March. 1. Hibernating animals: The liver cells contain much glycogen and large mitochondria, poorly developed ER, relatively well developed Golgi apparatus, various amounts of lysosomes. Most of the bile canaliculi are closed. 2. Non-hibernating, starving animals: 24 hours after arousal the glycogen and lipid droplets disappear from the liver cells, the mitochondria swell slightly. There is no essential alteration in the ER. In the liver cells of the starving animals numerous autophagic vacuoles appear including disintegrated cytoplasmic components. 3. Non-hibernating force-fed animals: They were given food rich in fat and protein, containing sugar and a drug increasing bile secretion. Within an hour, glycogen and lipid reappears in the liver cells, autophagic vacuoles disappear, mitochondria become larger. After feeding, the Golgi apparatus hypertrophies considerably, many bile canaliculi open, the number of the peribiliary lysosomes decreases.This paper is dedicated to Professor W. Bargmann on the occassion of his 60th birthday.     

Fine structure of antheridia and oogonia of Phytophthora macrospora,the downy mildew fungus of rice plants

In the present paper fine structure of antheridium and oogonium ofPhytophthora macrospora (Sacc.)S. Ito etI. Tanaka, the downy mildew fungus of rice plants was discussed.Before the fertilization some nuclei and a large number of mitochondria were scattered in the cytoplasm of the antheridium. Many lipid granules were observed in the peripheral region, but vacuoles did not appear at this stage of antheridium. Many mitochondria were associated in the neighborhood of the fertilization pore. The wall at the pore was very thin, but the wall surrounding the pore was slightly swollen towards the inside.In the oogonium, many nuclei, mitochondria and cytoplasmic matrix were observed at the peripheral part. A large number of lipid granules was found in the oogonium, but they were more numerous in the peripheral region. The vacuoles developed as the oogonium matured. They were enveloped by tonoplast and contained vacuolar matrix. Many electron dense granules were in contact with the tonoplast or free in the vacuoles, and they were larger in the central part. As stated above, wall at the fertilization pore was thin. However, the oogonial wall surrounding the pore swelled protruding into the oogonium. An electron-dense layer was recognized between the antheridial and oogonial wall, and the walls of both the organs were closely in contact with each other.Contribution No. 252.     

THE MORPHOLOGY AND FINE STRUCTURE OF CERATIUM HIRUNDINELLA (DINOPHYCEAE)1

Some aspects of thecal morphology in Ceratium hirundinella are redescribed by means of scanning electron microscopy. The internal anatomy of the cell has been clearly revealed for the first time in serial sections examined by transmission electron microscopy. An unusual sulcus, termed the sulcul aperture, has been found to open into a large ventral chamber which extends toward the center of the cell. The 2 flagella are inserted at one side of the ventral chamber. The ultrastructure of the flagellar canals and root systems arc described in detail. A microtubular strand has been found to run from near the nucleus to near the flagellar bases but not to conned with either. The fine structure of the theca, trichocysts, nucleus, and chloroplasts is basically similar to that of other dinoflagellates. Food vacuoles are reported. These contain material thought to be undergoing digestion. It is suggested that the food particles enter the cell by way of the ventral chamber, which is lined only by membranes, unlike the rest of the cell, which is covered by thick plates. The new findings are discussed in relation to the earlier work on the species and to the fine structure of oilier dinoflagellates.     

MUCILAGE-PRODUCING CELLS IN THE SEED COAT OF PLANTAGO OVATA: DEVELOPMENTAL FINE STRUCTURE

As the ovule of Plantago ovata matures into a seed its epidermal cells are transformed from undifferentiated parenchyma to thin-walled containers of almost pure mucilage. During this process the volume of the cells increases 60–80 fold, and the protoplast degenerates to a remnant. Rapid cell expansion begins with pollination and is accompanied by an increase in the size of the nucleus and nucleolus, a change in the random arrangement of ribosomes, a decrease in the thickness of cell walls, and synthesis of starch. Deposition of mucilage inside vacuoles and between the plasma membrane and cell wall accompanies a marked increase in the number and size of Golgi vesicles. Histochemical evidence using the thiocarbohydrazide-osmium vapor method shows polysaccharide to be present within Golgi vesicles while they are still attached to the Golgi apparatus. Mucilage deposition is associated with further cell expansion, separation of the protoplast from the cell wall, fusion of vacuoles and extra protoplasmic space, and the disappearance of starch.     

Studies of the fine structure of ovarian interstitial tissue

Summary The fine structure of ovarian steroid-producing cells of the domestic fowl as seen after administration of steroids is described. Diaethylstilboestrol, estradiol and hydroxyprogesterone were given as intramuscular injections for a 28-days period. The main cytoplasmic changes of the steroid-producing cells were an increase in the number and the size of lipid droplets due to a possible expansion of the endoplasmic reticulum and a confluence of the smooth endoplasmic reticulum and the lipid droplets. The mitochondria became paler, markedly reduced in size and number, and contained less cristae. The Golgi apparatus became smaller and tended to disappear. The most prominent nuclear changes were irregular outline of the nuclear membrane, shrinkage of the nucleus, formation of pseudo-inclusions, decreased number of nuclear bodies with a tendency to vacuolization and small nucleoli. These alterations have never been demonstrated in ovarian steroid-producing cells before, and they are consistent with atrophic changes observed in steroid-producing cells in other organs. The present study substantiates the view that the thecal gland must be concerned with endocrine function. The possible functions of the different cell types of the thecal gland are discussed.