Electron-dense Layer Research Articles

Histological characteristics of the oocyte chorion in wild post-spawning and artificially matured Japanese eels Anguilla japonica

To describe the histological characteristics of the oocyte chorion in wild adult and artificially matured Japanese eels, we investigated changes in chorion thickness during artificially induced oogenesis and compared the chorion thickness and ultrastructure between wild and artificially matured eels. In artificially maturing eels, the chorion thickness and volume increased significantly with increasing follicle diameter, peaking at approximately 450 µm; beyond this point, the chorion thinned significantly, whereas there were no significant changes in volume. A significant positive correlation was observed between the number of salmon pituitary extract (SPE) injections and chorion thickness. In wild post-spawning adult eels, chorion thickness varied among individuals, and two had chorions that were significantly thinner than those of artificially matured eels. Ultrastructural examination revealed electron-dense layers in the chorions of wild post-spawning adult eels, as was seen in artificially matured eels. This result is inconsistent with our hypothesis that the formation of an electron-dense layer is unique to artificially maturing eels due to repeated SPE injections. These results suggest that the formation cycle of the chorion might be affected by SPE injections in artificially maturing eels, whereas that of wild eels might be synchronized with behavioral and/or environmental fluctuations that occur during the oceanic spawning migration.

Ultrastructure and chemical composition of the proboscis hooks of Acanthocephalus lucii (Müller, 1776) (Acanthocephala: Palaeacanthocephala) using X-ray elemental analysis

The ultrastructure and chemical composition of the proboscis hooks and surrounding tegument of Acanthocephalus lucii (Müller, 1776), a parasite of European perch, Perca fluviatilis Linnaeus, were examined using scanning (SEM) and transmission (TEM) electron microscopy and X-ray microanalysis (EDXA). The blade of middle hooks consists of three layers: an outer homogeneous layer, an inner heterogeneous layer and a central core. TEM observation revealed the presence of hollow tubes, which spaced the central core; fibrous inner hook layer surrounded by an electron-dense margin and the basal tegumental layer filled with electron-dense bodies and outer layer. We found for the first time that the so-called 'epidermal covering' surrounding of the exposed hook blade (outer hook layer) is a modified striped portion of the tegumental layer and there are no special contact sites between these two morphologically different structures, i.e. striped layer of the syncytial tegument and following proper outer hook layer, which is a homogeneous, moderately electron-dense layer of -0.3 μm in thickness. The hook root is embedded into subtegumental fibrous layer. X-ray microanalysis of both the surface and internal parts of A. lucii hooks demonstrated the presence of calcium, magnesium, phosphorus and sulphur. The highest concentration of sulphur was recorded at the tip of hooks, whereas the middle part of the hooks was most rich in calcium, phosphorus and magnesium. The proximal part of the hooks contained lower concentrations of sulphur, calcium and phosphorus. In the proboscis tegument, only two elements, calcium and silicon, were found. The differences observed in the chemical composition of the hook 'epidermal covering' and the proboscis tegument support our ultrastructural findings that the hook tegumental covering is a modified structure compared with that of the general proboscis tegument.

Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM’s from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

Brine assemblages of ultrasmall microbial cells within the ice cover of Lake Vida, Antarctica.

The anoxic and freezing brine that permeates Lake Vida's perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.

Porphyromonas gingivalis Outer Membrane Vesicles Exclusively Contain Outer Membrane and Periplasmic Proteins and Carry a Cargo Enriched with Virulence Factors

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Eimeria arloingi: Further studies on the development of some endogenous stages

The present study investigated the ultrastructural characteristics of gamogony and oocyst wall formation of Eimeria arloingi in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1×103 and 1×105 sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42days postinoculation (DPI), 1 kid from each group was necropsied for ultrastructural studies. Transmission electron microscopy was used to screen for the presence of developmental stages of the parasite. All stages of microgametocyte and macrogametocyte developments and also oocyst wall formation were observed from 7 to 42DPI. Different stages of schizigony accompanied by marked proliferation of endoplasmic reticulum, mitochondria and several granular dividing nuclei were diagnosed in the affected epithelial cells. Young microgamonts were recognizable by an electron lucent parasitophorous vacuole and several dividing nuclei with prominent nucleolar and peripheral chromatin in the cytoplasm. At a later stage, the nuclei began to elongate and a single mitochondrion and two basal bodies were observed in close proximity nucleus. These bodies eventually protruded from the surface of the gametocyte and formed two flagellar structures. Up to 80–120 microgametes were produced per microgamont. Macrogamonts were recognized by the presence of wall-forming bodies of types 1 and 2. Electron lucent WFB2 appeared earlier than the electron denser WFB1 during the process of macrogametogenesis. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface to form an electron dense layer. The WFB2 appeared, subsequently, to give rise to the electron lucent inner layer. WFB1 plays a major role in oocyst wall formation.

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.

Fine morphology of the jaw apparatus of Puncturella noachina (Fissurellidae, Vetigastropoda)

Jaws of various kinds occur in virtually all groups of Mollusca, except for Polyplacophora and Bivalvia. Molluscan jaws are formed by the buccal epithelium and either constitute a single plate, a paired formation or a serial structure. Buccal ectodermal structures in gastropods are rather different. They can be nonrenewable or having final growth, like the hooks in Clione (Gastropoda, Gymnosomata). In this case, they are formed by a single cell. Conversely, they can be renewable during the entire life span and in this case they are formed by a set of cells, like the formation of the radula. The fine structure of the jaws was studied in the gastropod Puncturella noachina. The jaw is situated in the buccal cavity and consists of paired elongated cuticular plates. On the anterior edge of each cuticular plate there are numerous longitudinally oriented rodlets disposed over the entire jaw surface and immersed into a cuticular matrix. The jaw can be divided into four zones situated successively toward the anterior edge: 1) the posterior area: the zone of formation of the thick cuticle covering the entire jaw and forming the electron-dense outer layer of the jaw plate; 2) the zone of rodlet formation; 3) the zone of rodlet arrangement; and 4) the anterior zone: the free scraping edge of the plate, or the erosion zone. In the general pattern of jaw formation, Puncturella noachina resembles Testudinalia tessulata (Patellogastropoda) studied previously. The basis of the jaw is a cuticular plate formed by the activity of the strongly developed microvillar apparatus of the gnathoepithelium. However, the mechanism of renewal of the jaw anterior part in P. noachina is much more complex as its scraping edge consists not just of a thick cuticular matrix rather than of a system of denticles being the projecting endings of rodlets.

Aedes aegypti midgut remodeling during metamorphosis

The Aedes aegypti midgut is restructured during metamorphosis; its epithelium is renewed by replacing the digestive and endocrine cells through stem or regenerative cell differentiation. Shortly after pupation (white pupae) begins, the larval digestive cells are histolized and show signs of degeneration, such as autophagic vacuoles and disintegrating microvilli. Simultaneously, differentiating cells derived from larval stem cells form an electron-dense layer that is visible 24h after pupation begins. Forty-eight hours after pupation onset, the differentiating cells yield an electron-lucent cytoplasm rich in microvilli and organelles. Dividing stem cells were observed in the fourth instar larvae and during the first 24h of pupation, which suggests that stem cells proliferate at the end of the larval period and during pupation. This study discusses various aspects of the changes during midgut remodeling for pupating A. aegypti.

Morphological and Ultrastructural Characteristics of the Chorion of Cimex lectularius Linnaeus (Hemiptera: Cimicidae)

Bed bugs, Cimex lectularius Linnaeus, are increasingly reported as pests in hotels, apartments and homes worldwide. Although limited ultrastructural analysis of bed bug nymphs and adults has been performed, little is known about the ultrastructure of bed bug eggs. This study, using scanning and transmission electron microscropy, revealed several unique structural aspects of bed bug eggs. Chorionic projections form a polygonal pattern covering the egg surface. A complex operculumcollar structure is found on the anterior end of the egg, and the inner surface of the collar has three distinct regions, a smooth outer rim, a porous palisade layer and a porous ribbed layer. The outer surface of the collar is smooth with an operculum fitting into the collar. Large and distinctly ridged polygons cover the surface of the operculum. The polygons around the perimeter of the operculum have a rugose surface; the polygons adjacent to the rugose ones display spheres on the surface; and the remaining polygons have a smooth surface. The lateral margin of the operculum has a porous rugose surface with ridges and the ventral inner surface is smooth. Internally, the chorion is made up of three distinct divisions, the outer electron-dense layer with round electron opaque areas near the surface, a middle layer which is less electron-dense and the innermost layer of porous struts creating distinct spaces. Three distinct layers are found in the operculum, an outer porous layer, then an electron-dense layer, and an inner layer of porous struts and spaces. These findings constitute the first detailed description of the bed bug egg using electron microscopy.

Development of an approach to analyze the interaction between Nosema bombycis (microsporidia) deproteinated chitin spore coats and spore wall proteins

Nosema bombycis is an obligate intracellular parasite of the Bombyx mori insect. The spore wall of N. bombycis is composed of an electron-dense proteinaceous outer layer and an electron-transparent chitinous inner layer, and the spore wall is connected to the plasma membrane. In this study, the deproteinated chitin spore coats (DCSCs) were acquired by boiling N. bombycis in 1M NaOH. Under a transmission electron microscope, the chitin spore coat resembles a loosely curled ring with strong refractivity; organelles and nuclei were not observed inside the spore. The anti-SWP25, 26, 30 and 32 antibodies were used to detect whether spore wall proteins within the total soluble and mature spore proteins could bind to the DCSCs. Furthermore, a chitin binding assay showed that within the total soluble and mature spore proteins, the SWP26, SWP30 and SWP32 spore wall proteins, bound to the deproteinated chitin spore coats, although SWP25 was incapable of this interaction. Moreover, after the DCSCs were incubated with the alkali-soluble proteins, the latter were obtained by treating N. bombycis with 0.1M NaOH. Following this treatment, SWP32 was still capable of binding the DCSCs, while SWP26 and SWP30 were unable to bind. Collectively, the DCSCs are useful for investigating the arrangement of spore wall proteins, and they shed light on how the microsporidia spore wall is self-assembled.

The vacuoles containing multivesicular bodies: a new observation in interaction between Ustilago esculenta and Zizania latifolia

Ustilago esculenta infects Zizania latifolia plants and induces swelling on their upper culms. As an edible aquatic vegetable, these swellings are called jiaobai (white bamboo). The process of fungal development in Huijiao (grey bamboo) plants from seedlings to upper culm swelling was investigated using knife cut tissues, light and electron microscopy. All Huijiao plants were relatively taller than all jiaobai plants and in knife-cut sections sori development differed after culm swelling in Huijiao plants. Light microscope examination show that hyphae were both intracellular and intercellular, forming hyphal aggregations, and were more abundant in nodes than in internodes. Rapid increase of hyphal aggregations occurred 15 days before culm swelling, causing the death of abundant host cells, and hyphal aggregations differentiated into sori initiates. Ultrastructural observation showed that intracellular hyphae at the interface of fungus-host interaction were encased in a sheath with outer electron-opaque and inner electron-dense layer. Scale-like material and membrane-bounded vesicles were often present in an outer electron-opaque matrix of the sheath. Vacuoles, lipid globules (LGs) and multivesicular bodies (MVBs) were occasionally seen in these fungal cells. In summer, abundant electron-dense MVBs, LGs and fewer vacuoles containing MVBs were present in a fungal cell. As the temperature decreased, some MVBs became electron-opaque, some internal vesicles were also electron-opaque, and subsequently, the MVBs disappeared, leaving irregular remnant materials in vacuoles. During the rapid increase in hyphal aggregations, the irregular remnants of material within the vacuoles disappeared and LGs were rare. At the beginning of sori development, LGs once again became abundant in the cells of hyphal aggregation. The process of sorus and teliospore development was observed systematically and relationships between each developmental stage examined from knife cut tissues, and cytological and ultrastructural sections. Sorus development occurred with the commencement of upper culm swelling and differed in the same tissues. Teliospore development was also uneven in a sorus. The inner region of a sorus developed earlier than the outer part.

Corpora amylacea in gastrointestinal leiomyomas: a clinical, light microscopic, ultrastructural and immunohistochemical study with comparison to hyaline globules

ContextCorpora amylacea (CA) are inclusions with starch-like composition that occur in various conditions. We have observed CA in gastrointestinal leiomyomas (GILM) and hypothesised that they differ from intracytoplasmic hyaline globules...

Ultrastructural study on dynamics of lipid bodies and plastids during ripening of chili pepper fruits

Dynamics of lipid bodies and plastids in chili pepper fruits during ripening were investigated by means of transmission electron microscopy. Mesocarp of chili pepper fruits consists of collenchyma, normal parenchyma, and huge celled parenchyma. In mature green fruits, plastids contain numerous thylakoids that are well organized into grana in collenchyma, a strikingly huge amount of starch and irregularly organized thylakoids in normal parenchyma, and simple tubes rather than thylakoids in huge celled parenchyma. These morphological features suggest that plastids are chloroplasts in collenchyma, chloroamyloplasts in normal parenchyma, proplastids in huge celled parenchyma. As fruits ripen to red, plastids in all cell types convert to chromoplasts and, concomitantly, lipid bodies accumulate in both cytoplasm and chromoplasts. Cytosolic lipid bodies are lined up in a regular layer adjacent to plasma membrane. The cytosolic lipid body consists of a core surrounded by a membrane. The core is comprised of a more electron-dense central part enclosed by a slightly less electron-dense peripheral layer. Plastidial lipid bodies in collenchyma, normal parenchyma, and endodermis initiate as plastoglobuli, which in turn convert to rod-like structures. Therefore, plastidial lipid bodies are more dynamic than cytosolic lipid bodies. Both cytosolic and plastidial lipid bodies contain rich unsaturated lipids.

Quantitative Silencing of EGFP Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol

Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N4,N9-diacyl spermine conjugate, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N4,N9-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.

Spermatological characteristics ofElstia stossichianum(Digenea, Mesometridae) from the intestine of the cow bream (Sarpa salpa) off Dakar, Senegal

The current study was designed to increase the spermatological ultrastructural database on Digenea, thus contributing to the future establishment of phylogenetic relationships within this group based on ultrastructural characteristics of both spermiogenesis and spermatozoa. Spermiogenesis in Elstia stossichianum begins with the formation of a differentiation zone containing two centrioles, two striated rootlets, a nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. Each centriole develops into a free flagellum growing orthogonally to the median cytoplasmic process. Proximo-distal fusion of the flagella with the median cytoplasmic process occurs after flagellar rotation. Both nucleus and mitochondria migrate before the complete proximo-distal fusion of both flagella with the median cytoplasmic process. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon. The mature spermatozoon of E. stossichianum exhibits two axonemes of different length of the 9+'1' trepaxonematan pattern, a nucleus, a mitochondrion, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, a lateral expansion, spine-like bodies, cytoplasmic ornamented buttons and granules of glycogen. The formation of cytoplasmic ornamented buttons during the final stages of spermiogenesis is reported here for the first time.

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9+‘1’ trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.

Ultrastructural Evidence of Oogenesis of the Rusty Parrotfish, Scarus ferrugineus (Teleostei: Scaridae)

Ultrastructural study of oogenesis of the Scarus ferrugineus was carried out during the period of oocyte growth, particularly the changes in nuclear morphology, cytoplasmic organelles, and wall formation. Materials produced in the nucleus and transported to the cytoplasm known as nuage were observed in the oogonia and primary oocytes at premeiotic and perinucleolar stages. This material gives rise to germ plasma in the growing oocytes. In the perinucleolar oocyte, the cytoplasm appears loaded with free ribosomes and dense bodies. Short strands of endoplasmic reticulum (ER), perinuclear mitochondrial ring, and a simple layer of flattened granulosa cells were also detected. Protrusion of short buds from oocyte and granulose surfaces to the perioocyte space was clearly detected in the oocyte. From the early vitellogenic stage (yolk vesicle oocyte) the vitelline envelope was observed as a single electron-dense mesh pattern layer, becoming thicker during the vitellogenic stage. The granulosa cells (GCs) become organelle-rich with elongate mitochondria, free ribosomes, dilated tubular RER, and a Golgi system. Both GCs and thecal cells (TCs) show ultrastructural steroidogenic features, suggesting their primary roles in the production and secretion of steroid hormones at this stage of ovarian development. Remarkable ultrastructural changes were shown in vitellogenic oocytes including increase in RER, proliferation of mitochondria, and protrusion of long microvilli from oocyte and granulosa cells into pore canals of the vitelline membrane. The microvilli withdraw by the end of yolk deposition and the granulosa cells dissociate in fully grown oocytes. In atretic oocytes, the vitelline membrane homogenized and fragmented. Also, GCs, and maybe TCs, proliferated, hypertrophied, and phagocytosed the oocyte remnants forming temporary structure. Degeneration of previtellogenic oocytes prior to sex change was described and discussed.

Development of thallus axes in Usnea longissima (Parmeliaceae, Ascomycota), a fruticose lichen showing diffuse growth

While cell wall thickening in plants is generally associated with tissue maturation, fungal tissues in at least two lichens continue to grow extensively while accumulating massively thickened cell walls. We examined Usnea longissima to determine how diffuse growth shapes morphological and anatomical development of thallus axes and how the highly thickened cell walls of the central cord behave in diffuse growth. Fresh material was examined with light and epifluorescence microscopy and conventional and low-temperature SEM. Fixed material was embedded in Spurr's resin, microtome-sectioned, and examined with TEM and light microscopy. Main axes consisted essentially of bare medullary cord tissue; their characteristic morphology developed by destruction of the overlying cortex and consequent stimulation of lateral branch formation. Fungal cells of the cord tissue continually deposited wall layers of electron-transparent substances and layered, electron-dense materials that include UV-epifluorescent components. Discontinuities were evident in the outermost layers; new branch cells grew through wall materials accumulated by older neighboring cells. Sustained diffuse growth of cord tissue in U. longissima underlies the structural transformation of a corticated thallus branch into a long axis. In the cord tissue, diffuse growth may be responsible for the increasingly disrupted appearance of the older, electron-dense cell wall layers, while new wall materials are laid down adjacent to the protoplast. Cell and tissue development appeared comparable to that observed previously in Ramalina menziesii, although accumulation of wall material was somewhat less extensive and with a greater proportion of electron-dense/UV-epifluorescent components.

Zebra mussel adhesion: Structure of the byssal adhesive apparatus in the freshwater mussel, Dreissena polymorpha

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well.