Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Ultrastructural studies of the nucleoids of the pleomorphic forms of Chlamydia psittaci 6BC: a comparison with bacteria.

The nucleoids of the various pleomorphic forms of Chlamydia psittaci have been examined by direct observation of infected cells and by observations on isolated particles. The fixation and staining methods used were the same as those routinely used for the examination of bacteria to facilitate the comparison of chlamydial fine structure with that of bacteria. The nucleoids of reticulate bodies were composed of fine fibrils which extended throughout these particles. The nucleoids of intermediate bodies are characterized by an electron-dense mass with which the fibrous elements are associated in a structurally coherent manner. As condensation of the intermediate bodies proceeds, the electron-dense mass becomes eccentrically located and the fibers form a distinct radiating structure. Large elementary bodies have a few fibers associated with their condensed electron-dense nucleoids but the more condensed mature elementary bodies have a very discrete and homogeneous electron-dense nucleoid which is separated from the cytoplasmic elements of these particles by a very distinct electron-transparent space. These highly condensed elementary body nucleoids are usually ovoid, but may be elongated or irregular, and a small number of these structures react very strongly with ruthenium red. While the nucleoid structure of reticulate bodies resembles that of the bacterial cell, both the condensation process and the nucleoid morphologies which result from it in intermediate and elementary bodies have no parallels among the bacteria. Thus we conclude that major differences in nucleoid organization exist between the chlamydia and the bacteria.     

Cytochemical localization of glycogen in Chlamydia trachomatis inclusions.

The origin and distribution of glycogen in inclusions of Chlamydia trachomatis were demonstrated with silver proteinate stain for electron microscopy. Glycogen particles were detected in all developmental stages of C. trachomatis, as well as free in the inclusions. Intrachlamydial glycogen was most common in elementary bodies but was also detected in intermediate forms and reticulate bodies (RBs). Abnormal divisions and breakdown of cytoplasmic membranes were common in RBs. Cytoplasmic contents, including glycogen particles, were released into the inclusions after rupture of the outer membranes of abnormal RBs and intermediate forms. From these observations, we conclude that glycogen in inclusions of C. trachomatis originates in the organisms themselves.     

Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp

Significant differences in cysteine-containing proteins and detergent-related solubility properties were observed between outer membrane protein complexes of reproductive (reticulate body) and infective (elementary body) forms of Chlamydia psittaci (6BC). Elementary bodies harvested at 48 h postinfection possessed a 40-kilodalton major outer membrane protein and three extraordinarily cysteine-rich outer membrane proteins of 62, 59, and 12 kilodaltons, all of which were not solubilized by sodium dodecyl sulfate in the absence of thiol reagents. Intracellularly dividing reticulate bodies harvested at 21 h postinfection were severely deficient in the cysteine-rich proteins but possessed almost as much major outer membrane protein as did the elementary bodies. Most of the major outer membrane protein of reticulate bodies was solubilized by sodium dodecyl sulfate and was present in envelopes as monomers, although a proportion formed disulfide-cross-linked oligomers. By 21 to 24 h postinfection, reticulate bodies commenced synthesis of the cysteine-rich proteins which were found in outer membranes as disulfide-cross-linked complexes. The outer membranes of reticulate bodies of Chlamydia trachomatis (LGV434) also were found to be deficient in cysteine-rich proteins and to be more susceptible to dissociation in sodium dodecyl sulfate than were outer membranes of elementary bodies.     

Comparative midgut ultrastructure of unfed larvae and adult mites of Platytrombidium fasciatum (C.L. Koch, 1836) and Camerotrombidium pexatum (C.L. Koch, 1837) (Acariformes: Microtrombidiidae)

The midgut of unfed larvae and adult mites of Platytrombidium fasciatum (C.L. Koch, 1836) and Camerotrombidium pexatum (C.L. Koch, 1937) (Acariformes: Microtrombidiidae) was investigated by electron microscopy. The sac-like midgut occupies the entire body volume, ends blindly and is not divided into functionally differentiated diverticula or caeca. The midgut walls are composed of one type of digestive cell that greatly varies in shape and size. In larvae, the lumen of the midgut is poorly recognizable and its epithelium is loosely organized, although yolk granules are already utilized. In adults, the midgut forms compartments as a result of deep folds of the midgut walls, and the lumen is well distinguished. The epithelium is composed of flat, prismatic or club-like cells, which may contain nutritional vacuoles and residual bodies in various proportions that depend on digestive stages. In both larvae and adult mites, parts of cells may detach from the epithelium and float within the lumen. The cells contain a system of tubules and vesicles of a trans-Golgi network, whereas the apical surface forms microvilli as well as pinocytotic pits and vesicles. Lysosome-like bodies, lipid inclusions and some amount of glycogen particles are also present in the digestive cells. Spherites (concretions) are not found to be a constant component of the digestive cells and in adult mites occur for the most parts in the midgut lumen.     

Preparation and Chemical Composition of the Cell Membranes of Developmental Reticulate Forms of Meningopneumonitis Organisms

The outer limiting membranes of developmental reticulate forms of the meningopneumonitis organism were purified by a combination of differential centrifugation, trypsin digestion, and sodium dodecyl sulfate treatment, and their physical and chemical properties were compared with those of outer envelopes of mature dense forms of this organism. Reticulate bodies were easily disrupted by short periods of sonic treatment and were lysed by trysin digestion, in contrast to the dense bodies which were resistant to these treatments. In electron micrographs, reticulate body membranes were seen as very thin, flattened structures, whereas dense-body envelopes showed folding rigid membranes. The results of chemical fractionation of (32)P-labeled purified preparations indicated that reticulate body membranes have smaller amounts of phospholipid, and are more dense than cell walls of the mature forms. The analysis of amino acid composition of reticulate body cell membranes showed that they do not contain cystine or methionine, both of which were found in cell walls of dense bodies. These results clearly show that there are significant differences in the chemical and physical properties of the outer envelopes of the developmental and mature forms of this organism.     

Ultrastructural studies of Chlamydia psittaci 6BC in situ in yolk sac explants and L cells: a comparison with gram-negative bacteria.

Chlamydia psittaci (6BC) was grown in yolk sac explants and in L cells and fixed by perfusion in situ to provide undamaged material for comparison with gram-negative bacteria. Reticulate, intermediate, and elementary bodies were all seen to lack a well-defined periplasmic space; intermediate and elementary bodies showed condensations of the nucleoid which differ from common bacterial configurations; and the cytoplasm of highly condensed elementary bodies was much more electron dense than that of the gram-negative bacteria, while retaining its basically particulate nature. These important morphological distinctions are interpreted as reflections of a significantly different cellular level of organization in these two groups of organisms. No important morphological differences were noted in comparisons of the chlamydial particles grown in the two different host systems.     

Immunocytochemical localization of a urate oxidase immunoreactive protein in the plasma membranes and membranes of the secretory/endocytic compartments of digestive gland cells of the mussel Mytilus galloprovincialis

The subcellular compartmentalization of urate oxidase (UOX) in the digestive glands of mussels, Mytilus galloprovincialis Lmk, was studied by means of immunoblotting and immunocytochemistry, using an antibody raised in rabbit against rat liver UOX. Western blot analysis of subcellular fractions revealed an immunoreactive polypeptide with a molecular weight similar to the corresponding mammalian hepatic protein. This crossreactive polypeptide of 32 kDa was particle-bound yet not peroxisome-associated. In paraffin sections the antiserum specifically labeled the plasma membrane of the digestive gland epithelial cells and discrete regions within the perinuclear and apical portions of the digestive tubules and duct cells. By electron microscopy gold particles representing antigenic sites were found on the microvilli and the lateral plasma membrane as well as the membranes of the secretory/ endocytic compartments, that is, the Golgi complex, secretory and some endocytic vesicle membranes. Since the peroxisomal UOX-antibody exhibits a comparable immunoreactivity towards a urate-transporter channel protein in rat kidney proximal tubules and has been used for its molecular cloning (Leal-Pinto et al., 1997, J. Biol. Chem. 272, 617-625), we suggest that the membrane protein identified in mussel digestive glands could represent a homologous urate-transporter protein.     

Peroxisomes in the digestive diverticula of the bivalve mollusc Nucula sulcata

Summary Electron microscopy of the digestive diverticula of the protobranch bivalve, Nucula sulcata, revealed the presence of peroxisomes in the basal regions of the epithelial cells lining the main and secondary ducts, and in the digestive and secretory (basophil) cells of the tubules. Those in the secretory cells are elongate and somewhat flattened, while those of the other cell types have a spherical form. Two distinct types of nucleoid are normally present within the secretory cell peroxisomes, one compact, crystalline, and finely polytubular, the other comprising isolated secondary tubules arranged in a linear series across the width of the organelle. The peroxisomes of the digestive and duct cells contain coarsely polytubular cores arranged in two clusters orientated more or less at right angles; the duct cell peroxisomes may also contain a second nucleoid in the form of a compact finely polytubular core. Sections incubated in a medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide reveal an electron dense reaction product within the peroxisomes of all the cell types. Catalase is considered to be responsible for the reaction.     

Male and female reproductive morphology in the inseminating genus Astroblepus (Ostariophysi: Siluriformes: Astroblepidae)

The structure of the reproductive organs of males and females of four species of Astroblepus were studied by light microscopy and TEM. In females, the gonadal and urinary orifices are separated. The urinary pore opens at the tip of a urinary papilla. In males, the testes conform to the anastomosing tubular type. Spermatogenesis is partially cystic, with most spermatid stages taking place within the lumen of the seminiferous tubules. The sperm ducts pass through seminal vesicles. The main sperm ducts fuse and join the urinary duct to form a urogenital duct that passes through to the tip of an long urogenital papilla. A circularly arranged skeletal muscle at the base of the urogenital papilla probably serves as a sphincter. The spermatozoon is an anacrosomal introsperm with an elongate, conical nucleus and an elongate midpiece. The midpiece contains numerous fused mitochondria that form rings within the cytoplasmic collar, but no vesicles were observed. The flagellum has a classic 9 + 2 axoneme with the A-tubules of the peripheral doublets being electron-dense. Two lateral fins on the posterior part of the flagellum have electron-dense termini. Nuclear chromatin is highly condensed, and the two centrioles are oriented perpendicularly to one another and are contained within a nuclear fossa.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.     

Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes

A field study has been carried out to validate the measure of structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress. Previous laboratory studies demonstrated that the digestive lysosomal system of molluscs reponds to a variety of pollutants and to different stress situations by exhibiting significant changes in its structure. Mussels were collected monthly over 1 year at the Abra estuary (Bizkaia, Biscay Bay) from six sites with different degrees of pollution. The changes in the structure of the digestive lysosomes were quantified on cryostat sections of the digestive gland by means of automated image analysis. Four stereological parameters were recorded: lysosomal volume density, surface density, surface-to-volume ratio and numerical density. A seasonal pattern in the structure of the digestive lysosomes was evidenced with reduced volume, surface, size and numbers of lysosomes in winter-spring; increased volume, surface, size and numbers in summer and an intermediate situation in autumn. The structure of digestive lysosomes was also dissimilar among sites, the most significant differences being found between Plentzia (nonpolluted site) and Galea (polluted site). The digestive lysosomes of mussels collected from Galea were smaller and more abundant than in Plentzia's mussels in most sampling times. The basis of these differences are discussed to conclude that organic chemical pollution might be the cause for these specific changes which are different from the enlargement of digestive lysosomes described as a result of various sources of environmental stress. It is concluded that structural changes in the digestive lysosomes of sentinel mussels are sensitive to pollution-induced environmental stress even in the complex situation of the field where many factors may interact to affect the structure of the digestive lysosomal system.     

A study of the histology and morphology of the digestive tract of the common eel (Anguilla anguilla)

The anatomy and morphology of the digestive tract of the eel was examined using light and scanning electron microscopy. The oeosphagus showed complex longitudinal folding; stratified epithelium, columnar epithelium and goblet cells striated muscle fibres formed the thick muscular coat. The pneumatic duct entered the oesophagus anterior to the oesophagastric junction. The Y shaped stomach showed large well developed folds which decreased in size and number towards the pyloric sphincter. Columnar epithelium and gastric gland cells were present, the latter being absent from the pyloric region.
The intestine had a heavily thickened anterior region, signs of convulation were noted prior to the ileorectal valve. Intestinal folding showed a complex reticulate pattern with columnar epithelium and goblet cells present. The mucopolysaccharides were studied in the goblet and columnar cells throughout the regions of the gut. Lymphocytes and eosinophilic type cells were found in the connective tissue of the mucosa throughout the gut. The pancreas was a compact organ with few Islets of Langerhans, beta cells were peripherally situated and alpha cells centrally. The unilobular liver acted as a storage organ for oil and glycogen.     

Arylsulphatase activity associated with phenanthrene induced digestive cell deletion in the marine mussel Mytilus edulis

Summary The acid hydrolase arylsulphatase has been localized at the ultrastructural level in digestive cells of the marine musselMytilus edulis for control and phenanthrene-treated (200µg/l) animals. In untreated mussels the activity was generally restricted to the lysosomal—vacuolar system and the Golgi apparatus. It was associated with all types of vesicle, although not all individual vesicles were reactive. In heterolysosomes which were filled with precipitate the reaction product was most densely associated with the limiting membranes. Lipid inclusions commonly occurred in the digestive cells; these sometimes showed limited reaction for enzyme activity. The striking difference between normal and phenanthrene-treated samples was the presence in all treated animals of reaction product in the inter-cellular spaces and varying degrees of cytoplasmic activity in a number of digestive cells. This is interpreted as a sign of impending cell deletion. Sections for morphological examination showed evidence of increased digestive cell deletion in phenanthrene-treated mussels. The process results in release of membrane-bound bodies into the tubule lumen.     

DNA adducts as indicators of genotoxic exposure in indigenous and transplanted mussels, Mytilus edulis L. from Icelandic coastal sites

Indigenous mussels, Mytilus edulis, were collected at sites with supposed different amounts of pollution; Reykjavík harbour, Keflavík harbour, Grafarvogur and Hvalfj?rdur (reference), along the south-western coast of Iceland in March 2000. Mussels from Hvalfj?rdur and Reykjavík harbour were also collected in August the same year. Additionally, mussels were transplanted from the reference site to Reykjavík harbour for 6 weeks during both winter and summer for comparison. DNA adducts were analysed by 32P-post-labelling in gills and digestive gland. Highest adduct levels were found in gill tissue from indigenous mussels collected in Reykjavík harbour. Adduct levels in both tissues from mussels collected at the reference site were below or very close to the detection limit during winter, but seemed to increase a little during summer. Mussels from sites with supposed intermediate pollution had intermediate levels of DNA adducts in gills but did not differ from Reykjavík harbour in digestive gland. No increase in adduct levels was observed in mussels transplanted from the reference site to Reykjavík harbour, except for a slight increase in digestive gland during winter. This study shows that 32P-post-labelling analysis of DNA adducts is sensitive enough to be used on indigenous mussels from relatively pristine areas and that adduct levels are increased in harbours/urban sites. However, transplantation of mussels from a clean site to the harbour for 6 weeks did not result in increased adduct levels in gills, the tissue with the highest adduct levels. The results also indicate that seasonal variation in adduct levels may occur.     

Purification and Chemical Composition of Reticulate Bodies of the Meningopneumonitis Organisms

Reticulate bodies of the meningopneumonitis (MP) microorganism were purified from L cells 18 hr after infection by the combination of differential centrifugation in 30% sucrose solution and potassium tartrate density gradient centrifugation. It was ascertained by electron microscopy that purified preparations of reticulate bodies obtained were almost entirely free of host-cell components and of infectious elementary bodies of MP microorganisms. Purified reticulate bodies were easily disrupted by mechanical agitation, and it was observed in shadowed preparation that ribosome-like particles 15 mmu in diameter were scattered from broken reticulate bodies. In shadowed preparations, reticulate bodies were found to range in size from 1.0 to 1.6 mu in diameter, but in cross-section the range was 0.5 to 1.0 mu. In these preparations, the purified reticulate bodies were irregular in shape, round or oval, and were composed of rather homogenous, amorphous, or reticulate material with moderate density. Some particles exhibited a less-dense internal structure, in which a coarse fibrous reticulum was seen. Chemical fractionation of (32)P-labeled purified reticulate bodies showed that they contained three times more ribonucleic acid (RNA) than deoxyribonucleic acid, with the RNA being composed primarily of 21S, 16S, and 4S RNA. No infectivity of purified reticulate bodies could be demonstrated.     

Description and partial characterization of a new chlamydia-like microorganism

Abstract A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5–7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.     

Ultrastructure of bacterized and axenic trophozoites of Entamoeba histolytica with particular reference to helical bodies

Electron microscopy of bacterized and axenic trophozoites of Entamoeba histolytica showed only slight differences in ultrastructure between the two. As with other species of Entamoeba so far studied, this species lacks typical mitochondrial structures and formed endoplasmic reticulum. Dense clusters of glycogen particles are especially characteristic in axenic amebas. Microtubular structures 360 A in diameter appear randomly oriented in both bacterized and axenic trophozoites. Ribonucleoprotein (RNP) bodies are of two typical forms—elongate, parallel arrays of helices (the classical chromatoid bodies), and short helical fragments. Both kinds of helix show a recurring pitch angle of 68–80° and an over-all diameter of 480 A. RNP particles comprising the helices average 180 A in diameter. The longitudinal axes of adjacent helices are 440 A apart. Following RNase digestion of water-soluble methacrylate sections, helices show a core approximately 60 A in diameter. Short helices are also associated with digestive vacuoles. Free RNP particles per se are never seen within digestive vacuoles, but intact short helices are frequently detected closely associated with the external membrane of digestive vacuoles. In some cases, continuation of externally intact helical forms could be related to filamentous material within the vacuole. Acid phosphomonoesterase activity could be demonstrated within digestive vacuoles where deposition of reaction product is especially intense on the filamentous material.     

The effects of zinc oxide nanoparticles on the metallome in freshwater mussels

The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 μg/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn^2 +. For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn^2 +. Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn^2 +, suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn^2 + and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a “metallomic” approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.