Showing papers on "Ultrastructure published in 2020"

Light microscopy of proteins in their ultrastructural context.

Abstract: Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.

Ultrastructure of parenchyma cell wall in bamboo (Phyllostachys edulis) culms

Abstract: Parenchyma cell wall structure plays a crucial role in the growth and the mechanical properties of bamboo plants, with the secondary cell wall providing strength and rigidity. However, little is known about the ultrastructure of the parenchyma cell wall. The aim of this study was to characterize the anatomical structure of the parenchyma cell wall and determine how it contributes to great mechanical superiority of bamboo culm. We investigated the ultrastructure of the parenchyma cell wall using transmission electron microscopy and field-emission environmental scanning electron microscopy. The key results show that the secondary cell walls of ground and vascular parenchyma cells exhibited tight-loose (light-dark) alternating layers. The pit membrane of the ground parenchyma cells contained numerous pores, and that of vascular parenchyma cells contained some plasmodesmata. Secondary cell walls of most bamboo parenchyma cells contained seven sub-layers, with a maximum of eleven sub-layers of ground parenchyma cells and nine sub-layers of vascular parenchyma cells. The average thickness of ground parenchyma cell wall sub-layers was higher than that in vascular parenchyma cells. The pit membrane thickness of ground parenchyma cells was also higher than that of vascular parenchyma cells, but the diameter of the ground parenchyma cells was smaller than that of the vascular parenchyma cells. The extremely high flexibility of moso bamboo stem could be the consequence of the presence of secondary cell walls in parenchyma cells, and its ultrastructure.

Anatomical and ultrastructural responses of Hordeum sativum to the soil spiked by copper

Abstract: Effects of Cu toxicity from contaminated soil were analysed in spring barley (Hordeum sativum distichum), a widely cultivated species in South Russia. In this study, H. sativum was planted outdoors in one of the most fertile soils-Haplic Chernozem spiked with high concentration of Cu and examined between the boot and head emergence phase of growth. Copper toxicity was observed to cause slow ontogenetic development of plants, changing their morphometric parameters (shape, size, colour). To the best of our knowledge, the ultrastructural changes in roots, stems and leaves of H. sativum induced by excess Cu were fully characterized for the first time using transmission electron microscopy. The plant roots were the most effected, showing degradation of the epidermis, reduced number of parenchyma cells, as well as a significant decrease in the diameter of the stele and a disruption and modification to its cell structure. The comparative analysis of the ultrastructure of control plants and plants exposed to the toxic effects of Cu has made it possible to reveal significant disruption of the integrity of the cell wall and cytoplasmic membranes in the root with deposition of electron-dense material. The changes in the ultrastructure of the main cytoplasmic organelles-endoplasmic reticulum, mitochondria, chloroplasts and peroxisomes-in the stem and leaves were found. The cellular Cu deposition, anatomical and ultrastructural modifications could mainly account for the primary impact points of metal toxicity. Therefore, this work extends the available knowledge of the mechanisms of the Cu effect tolerance of barley.

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets

Abstract: Background State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure. Objectives Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. Methods We used serial block face-scanning electron microscopy images to render the 3D ultrastructure of α-granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. α-Granule compositional data were assessed by sequential, serial section cryo-immunogold electron microscopy and by immunofluorescence (structured illumination microscopy). Results and conclusions α-Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three-dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of α-granules came within ≤35 nm of each other, a distance associated in other cell systems with protein-mediated contact sites. Size and shape analysis of the 1488 α-granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all α-granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix.

Cells Undergo Major Changes in the Quantity of Cytoplasmic Organelles after Uptake of Gold Nanoparticles with Biologically Relevant Surface Coatings

Abstract: Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1-6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Abstract: Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum.

Abstract: Adaptation strategies in freezing resistance were investigated in Klebsormidium crenulatum, an early branching streptophyte green alga related to higher plants Klebsormidium grows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress Here, chilling and freezing induced alterations of the ultrastructure were investigated Control samples (kept at 20°C) were compared to chilled (4°C) as well as extracellularly frozen algae (-2 and -4°C) A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced Samples were then high pressure frozen and cryo-substituted for electron microscopy Control cells had a similar appearance in size and ultrastructure as previously reported While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (-2°C and -4°C) In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography Similar alterations/adaptations of the cell wall were not reported or visualized in Klebsormidium before, neither in controls, nor during other stress scenarios This indicates that the cell wall is reinforced by these additional wall layers during freezing stress Cells allowed to recover from freezing stress (-2°C) for 5 h at 20°C lost these additional cell wall layers, suggesting their dynamic formation The composition of these cell wall reinforcement areas was investigated by immuno-TEM In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography Measurements of the photosynthetic oxygen production showed an acclimation of Klebsormidium to chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles The cell wall reinforcement areas, together with the observed changes in organelle structure and distribution, are likely to contribute to maintenance of an undisturbed cell physiology and to adaptation to chilling and freezing stress

Reorganization of budding yeast cytoplasm upon energy depletion

Abstract: Yeast cells, when exposed to stress, can enter a protective state in which cell division, growth and metabolism are downregulated. They remain viable in this state until nutrients become available again. How cells enter this protective survival state and what happens at a cellular and subcellular level is largely unknown. In this study, we used electron tomography to investigate the stress-induced ultrastructural changes in the cytoplasm of yeast cells. After ATP depletion, we observed a significant cytosolic compaction and an extensive cytoplasmic reorganization, as well as the emergence of distinct membrane-bound and membrane-less organelles. By using correlative light and electron microscopy (CLEM), we further demonstrate that one of these membrane-less organelles is generated by the reversible polymerization into large bundles of filaments of the eukaryotic translation initiation factor 2B (eIF2B), an essential enzyme in the initiation of protein synthesis. The changes we observe are part of a stress-induced survival strategy, allowing yeast cells to save energy, protect proteins from degradation, and inhibit protein functionality by forming assemblies of said proteins.

Ultrastructure of the green alga Dunaliella salina strain CCAP19/18 (Chlorophyta) as investigated by quick-freeze deep-etch electron microscopy

Abstract: The single-celled green alga Dunaliella salina is a model system for studies on stress biology, in particular regarding secondary carotenoid accumulation. Under non-stress conditions the cells are green, but under abiotic stress the cells turn orange, because they switch their metabolism and accumulate β-Carotene in globules in the chloroplast. For the first time, Quick-freeze deep-etch electron microscopy was used to visualize cellular structures in green and orange cells of D. salina strain CCAP19/18. This allowed us to present an in-depth analysis of the cellular ultrastructure describing and comparing the features of the two cell types. Our images illustrate the presence of a pericellular matrix for this strain of D. salina. The pericellular matrix was spongy and strands of unknown material anchored it into the plasma membrane. The cytoplasm contained a variety of vesicles, vacuoles, and acidocalcisomes. We could show for strain CCAP19/18 that cytoplasmic lipid bodies were often in close proximity to and sometimes in contact with the outer chloroplast envelope membrane and with the endoplasmic reticulum. Major visible differences between green and orange cells were in the chloroplast: the orange cells have greatly reduced amounts of thylakoid membranes and greatly increased numbers of β-Carotene globules. We showed that the β-Carotene globules often made point contacts with thylakoid membranes, and frequently laid side-by-side along the thylakoid membrane surface, providing support to studies that indicated exchange of molecules between β-Carotene globules and thylakoid membranes. A novel finding was the β-Carotene globule duplets, suggesting intermediate stages in β-Carotene globule morphogenesis. Overall, the β-Carotene globules appear to be similar to plant plastoglobuli. We provide this description of the cellular ultrastructure features as a resource in the context of the recent publication of the genome of D. salina strain CCAP19/18 to expand on the knowledge regarding this novel reference strain.

Ultrastructural Analysis of Cells From Bell Pepper (Capsicum annuum) Infected With Bell Pepper Endornavirus.

Abstract: Endornaviruses include viruses that infect fungi, oomycetes, and plants. The genome of plant endornaviruses consists of linear ssRNA ranging in size from approximately 13-18 kb and lacking capsid protein and cell-to-cell movement capability. Although, plant endornaviruses have not been shown to cause detectable changes in the plant phenotype, they have been associated with alterations of the host physiology. Except for the association of cytoplasmic vesicles with infections by Vicia faba endornavirus, effects on the plant cell ultrastructure caused by endornaviruses have not been reported. Bell pepper endornavirus (BPEV) has been identified in several pepper (Capsicum spp.) species. We conducted ultrastructural analyses of cells from two near-isogenic lines of the bell pepper (C. annuum) cv. Marengo, one infected with BPEV and the other BPEV-free, and found cellular alterations associated with BPEV-infections. Some cells of plants infected with BPEV exhibited alterations of organelles and other cell components. Affected cells were located mainly in the mesophyll and phloem tissues. Altered organelles included mitochondrion, chloroplast, and nucleus. The mitochondria from BPEV-infected plants exhibited low number of cristae and electron-lucent regions. Chloroplasts contained plastoglobules and small vesicles in the surrounding cytoplasm. Translucent regions in thylakoids were observed, as well as hypertrophy of the chloroplast structure. Many membranous vesicles were observed in the stroma along the envelope. The nuclei revealed a dilation of the nuclear envelope with vesicles and perinuclear areas. The organelle changes were accompanied by membranous structure rearrangements, such as paramural bodies and multivesicular bodies. These alterations were not observed in cells from plants of the BPEV-free line. Overall, the observed ultrastructural cell alterations associated with BPEV are similar to those caused by plant viruses and viroids and suggest some degree of parasitic interaction between BPEV and the plant host.

Ultrastructure of Exospore Formation in Streptomyces Revealed by Cryo-Electron Tomography.

Abstract: Many bacteria form spores in response to adverse environmental conditions. Several sporulation pathways have evolved independently and occur through distinctive mechanisms. Here, using cryo-electron tomography (cryo-ET), we examine all stages of growth and exospore formation in the model organism Streptomyces albus. Our data reveal the native ultrastructure of vegetative hyphae, including the likely structures of the polarisome and cytoskeletal filaments. In addition, we observed septal junctions in vegetative septa, predicted to be involved in protein and DNA translocation between neighboring cells. During sporulation, the cell envelope undergoes dramatic remodeling, including the formation of a spore wall and two protective proteinaceous layers. Mature spores reveal the presence of a continuous spore coat and an irregular rodlet sheet. Together, these results provide an unprecedented examination of the ultrastructure in Streptomyces and further our understanding of the structural complexity of exospore formation.

Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High-Pressure Freezing/Freeze-Substitution.

Abstract: It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 μm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibril-associated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 2019. © 2019 American Association for Anatomy.

Does the frontal sensory organ in adults of the hoplonemertean Quasitetrastemma stimpsoni originate from the larval apical organ

Abstract: The apical organ is the most prominent neural structure in spiralian larvae. Although it has been thoroughly investigated in larvae of the class Pilidiophora in phylum Nemertea, studies on its structure in other nemertean larvae are limited. Most adult hoplonemertean worms have a frontal organ located in a position corresponding to that of the larval apical organ. The development and sensory function of the frontal organ has not been thoroughly characterized to date. The apical organ in the early rudiment stage of Quasitetrastemma stimpsoni larvae consists of an apical plate enclosed by ducts of frontal gland cells and eight apical neurons. The apical plate is abundantly innervated by neurites of apical neurons. During the late rudiment stage, the larval apical organ has external innervation from below by two subapical-plate neurons, along with 11 apical neurons, and its plate contains serotonin-like immunoreactive (5-HT-lir) cells. In the vermicular stage (free-swimming juvenile), the number of apical neurons is reduced, and their processes are resorbed. Serotonin is detected in the apical plate with no visible connection to apical neurons. In adult worms, the frontal organ has a small apical pit with openings for the frontal gland ducts. The organ consists of 8 to 10 densely packed 5-HT-lir cells that form the roundish pit. Although the ultrastructure of the Q. stimpsoni larval apical organ closely resembles that of the apical organ of Polycladida larvae, the former differs in the presence of flask-shaped neurons typical of Spiralia. Significant differences in the structure of the apical organs of hoplonemertean and pilidia larvae point to two different paths in the evolutionary transformation of the ancestral apical organ. Ultrastructural and immunoreactive analyses of the apical organ of a hoplonemertean larva in the late rudiment and vermicular stages and the frontal organ of the adult worms identified common morphological and functional features. Thus, we hypothesize that the larval apical organ is modified during morphogenesis to form the adult frontal organ, which fulfills a sensory function in the hoplonemertean worm. This unique developmental trait distinguishes the Hoplonemertea from other nemertean groups.

Physiological, anatomical and ultrastructural effects of aluminum on Styrax camporum , a native Cerrado woody species

Abstract: Styrax camporum Pohl (Styracaceae) is a woody species that grows on acidic soils from the Brazilian savanna with high aluminum (Al) saturation (m% > 50%), where it accumulates ~ 1500 mg Al per kg dry leaves Using nutrient solution, a previous study showed that 1480 μM Al causes toxicity symptoms, which raises the question whether less than 1480 μM Al could cause beneficial effects on this species Here, we checked possible altered gas exchange rates, damage to organelles in root tips and the association between Al exposure and mitochondria occurrence in cells of root tips, once organic acids from Krebs cycle exuded by the roots of this species when exposed to Al have been recently evidenced Five-month-old plants were grown in nutrient solution with 0, 740 and 1480 μM Al for 90 days Plants exposed to 1480 μM Al showed less developed root system, reduced plant height and low gas exchange rates in relation to those exposed to 0 and 740 μM Al, confirming that 1480 μM Al is toxic to S camporum However, plants exposed to 0 and 740 μM Al had similar number of leaves, plant height, root biomass, root length, total plant biomass and gas exchange rates, indicating that no beneficial effects from 740 μM Al could be noted on this species In plants exposed to 0 and 740 μM Al, mitochondria were noted at the root tip, while at 1480 μM Al these organelles were not evident due to the conspicuous vacuolation of root cells S camporum shows limited tolerance to Al in nutrient solution In addition, this species is not dependent on Al to grow and develop because the plants grew well under 0 and 740 μM Al

Glandular trichomes of Robinia viscosa Vent. var. hartwigii (Koehne) Ashe (Faboideae, Fabaceae)—morphology, histochemistry and ultrastructure

Abstract: Permanent glandular trichomes of Robinia viscosa var. hartwigii produce viscous secretion containing several secondary metabolites, as lipids, mucilage, flavonoids, proteins and alkaloids. Robinia viscosa var. hartwigii (Hartweg’s locust) is an ornamental tree with high apicultural value. It can be planted in urban greenery and in degraded areas. The shoots, leaves, and inflorescences of this plant are equipped with numerous persistent glandular trichomes producing sticky secretion. The distribution, origin, development, morphology, anatomy, and ultrastructure of glandular trichomes of Hartweg's locust flowers as well as the localisation and composition of their secretory products were investigated for the first time. To this end, light, scanning, and transmission electron microscopy combined with histochemical and fluorescence techniques were used. The massive glandular trichomes differing in the distribution, length, and stage of development were built of a multicellular and multiseriate stalk and a multicellular head. The secretory cells in the stalk and head had large nuclei with nucleoli, numerous chloroplasts with thylakoids and starch grains, mitochondria, endoplasmic reticulum profiles, Golgi apparatus, vesicles, and multivesicular bodies. Many vacuoles contained phenolic compounds dissolved or forming various condensed deposits. The secretion components were transported through symplast elements, and the granulocrine and eccrine modes of nectar secretion were observed. The secretion was accumulated in the subcuticular space at the trichome apex and released through a pore in the cuticle. Histochemical and fluorescence assays showed that the trichomes and secretion contained lipophilic and polyphenol compounds, polysaccharides, proteins, and alkaloids. We suggest that these metabolites may serve an important function in protection of plants against biotic stress conditions and may also be a source of phytopharmaceuticals in the future.

The Three-Dimensional Ultrastructure of the Human Alveolar Epithelium Revealed by Focused Ion Beam Electron Microscopy

Abstract: Thin type 1 alveolar epithelial (AE1) and surfactant producing type 2 alveolar epithelial (AE2) cells line the alveoli in the lung and are essential for normal lung function. Function is intimately interrelated to structure, so that detailed knowledge of the epithelial ultrastructure can significantly enhance our understanding of its function. The basolateral surface of the cells or the epithelial contact sites are of special interest, because they play an important role in intercellular communication or stabilizing the epithelium. The latter is in particular important for the lung with its variable volume. The aim of the present study was to investigate the three-dimensional (3D) ultrastructure of the human alveolar epithelium focusing on contact sites and the basolateral cell membrane of AE2 cells using focused ion beam electron microscopy and subsequent 3D reconstructions. The study provides detailed surface reconstructions of two AE1 cell domains and two AE2 cells, showing AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. Furthermore, we show reconstructions of a surfactant secretion pore, enlargements of the apical AE1 cell surface and long folds bordering grooves on the basal AE1 cell surface. The functional implications of our findings are discussed. These findings may lay the structural basis for further molecular investigations.

An Integrative Investigation of Parabistichella variabilis (Protista, Ciliophora, Hypotrichia) Including Its General Morphology, Ultrastructure, Ontogenesis, and Molecular Phylogeny.

Abstract: Hypotrichs are a highly differentiated and very diverse group of ciliated protists. Their systematics and taxonomy are challenging and call for detailed investigations on their general morphology, ultrastructure, ontogenesis, and molecular phylogeny. Here, a comprehensive study is conducted on a brackish water population of Parabistichella variabilis using light and electron microscopy and phylogenetic analyses based on small subunit ribosomal DNA sequence data. Its morphology, including the infraciliature, pellicle, nuclei, buccal seal, and extrusomes, is documented. The present findings indicate that in P. variabilis: (i) the cortical granules are extrusomes, which differ from those of other hypotrichs; (ii) the buccal seal is bounded by the plasma membrane and contains a single layer of longitudinal microtubules; (iii) two contractile vacuoles might be present rather than one; and (iv) the pharyngeal disks are bounded by a single membrane. Early-to-middle stages of ontogenesis are described for the first time, enabling the complete characterization of this process. Phylogenetic analyses indicate that Parabistichella variabilis is closely related to several species from different genera, such as Orthoamphisiella breviseries, Uroleptoides magnigranulosus, and Tachysoma pellionellum. However, ultrastructural and gene sequence data for more taxa are needed in order to resolve the systematics of Parabistichella.

The proteorhodopsins of the dinoflagellate Oxyrrhis marina : ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy

Abstract: At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles.

Enzymology, Histological and Ultrastructural Effects of Ar-Turmerone on Culex pipiens pallens Larvae.

Abstract: Our previous article demonstrated that ar-turmerone ((6S)-2-methyl-6-(4-methylphenyl)-2-hepten-4-one) extracted from Curcuma longa L. has a significant larvicidal activity against the fourth instar larvae of Culex pipiens pallens. To reveal the effects of ar-turmerone on C. pipiens pallens larvae, light microscopy and transmission electron microscopy were used to observe the histological and ultrastructure changes in muscle and digestive tissues of fourth instar larvae. It was also revealed by detecting the activity of the acetylcholinesterase (AChE) enzyme and three detoxifying enzymes, including carboxylesterase (CarE), glutathione-S-transferase (GST) and Cytochrome P450 monooxidases (P450). The observation under the light microscope showed that the larvae displayed a disruption of myofibril in ventral muscle cells, the disappearance of nucleolus in the malpighian tubule cells, and the exfoliation of the brush border in midgut epithelial cells, 24 h after treatment. The observation under the transmission electron microscope displayed disorganized Z-lines in the ventral muscle cells, and dissolved membrane of mitochondria, nuclear and endoplasmic reticulum in abdominal cells. The enzymatic activity results showed that ar-turmerone significantly increased the level of detoxifying enzymes, while the activity of AChE was not obviously affected. All the results suggest that the larvicidal mechanism of ar-turmerone is estimated to be stomach poison and the active sites might be the muscle and digestive tissues, and the mode of action of ar-turmerone may be unrelated to AChE.

Ultrastructure and cytochemistry of intrauterine embryonic and larval stages of Ityogonimus lorum (Digenea: Brachylaimidae) involving transitory development of ciliated miracidia

Abstract: Results of the present study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs while in the most posterior uterine regions of Ityogonimus lorum, a digenean parasite of an Iberian mole, Talpa occidentalis (Eulipotyphla, Talpidae). Using transmission electron microscopy (TEM), the ultrastructural characteristics of diverse cell types and their organelles of these developing embryos and fully formed miracidia within the eggshell were examined. The eggshell and embryonic envelopes are similar to those described previously by many authors for other digeneans. However, the developing miracidia are unique among previously described digeneans in possessing transitory cilia during larvigenesis, but completely lacking cilia in fully formed miracidium larvae. The evidence for completion of miracidial maturation in intrauterine eggs is based on the presence of the following structures: (1) transitional stage of ciliated differentiating miracidial epithelium; (2) apical and lateral glands, characteristic for digenean miracidia; and (3) fully developed germinative cells grouped together in the germinative sac localized in the posterior region of the miracidium. The protonephridial system with its characteristic flame cells and the nervous system with diverse types of neurons and nerve centers, which are characteristic for other digenean species reported until now, are absent from all these developmental stages of I. lorum. Based on these observations, we hypothesize that the life cycle of I. lorum is entirely terrestrial, involving passive transmission by ingestion of eggs containing unciliated miracidia to the first intermediate host.