合浦珠母贝鳃的显微与超微结构

合浦珠母贝(Pinctada fucata)是典型的滤食性瓣鳃类动物,也是我国重要的海水珍珠养殖贝类。本研究用光学显微镜、扫描电镜和透射电镜观察了合浦珠母贝鳃的显微和超微结构。结果表明,合浦珠母贝鳃结构属于异丝鳃型,左右两侧各2个鳃瓣,每个鳃瓣由内鳃瓣和外鳃瓣组成。鳃瓣由主鳃丝和普通鳃丝构成,主鳃丝在鳃瓣中主要起支架作用,每2根主鳃丝之间的9～12根普通鳃丝由"簇内连接"(intrabunchial junction)相连成簇。普通鳃丝之间通过"丝间连接"(interfilament junction)相连,丝间连接的上皮细胞与普通鳃丝的扁平细胞结构一样,为鳃的呼吸上皮。丝间连接的存在扩大了鳃的表面积,这种结构有助于进行气体交换。主鳃丝和普通鳃丝表面有前纤毛和侧纤毛,与食物运送和气体交换有关。普通鳃丝表面的纤毛为典型的"9+2"型微管结构。     

Rickettsiae in gill epithelial cells of the hard clam, Mercenaria mercenaria

Rickettsiae are found in the gill epithelium of the hard clam, Mercenaria mercenaria. The procaryotes occur free in the cytoplasm of the epithelial cells at the tip of the filament and in the more proximal cells that support the lateral J cilia. The fine structure of the organisms, showing rippled cell walls, is typical of the rickettsiae. The increasing size of the inclusion representing late phase growth often culminates in lysis of the host cell. Masses (Gram-negative, Feulgen-positive) in ova, similar to those observed in the gill epithelium, suggest that transovarian transmission may occur.     

Studies on the gill ciliation of the American oyster Crassostrea virginica (Gmelin)

Latero-frontal, para-latero-frontal, and frontal ciliary tracts on the gill filaments of Crassostrea virginica (Gmelin) were studied with light microscopy and scanning electron microscopy. Latero-frontal cirri are complex structures composed of varying numbers of paired cilia. The multiple pairs of cilia which constitute a single cirrus are closely appressed for a portion of their length; they then branch laterally from the central axis in a plume-like fashion. Latero-frontal cirri of adjacent gill filaments create a filtration sieve which should be capable of retaining particles smaller than 1 μm in diameter. Para-latero-frontal cilia are short, closely spaced cilia arranged as a staggered row along the frontal side of each tract of latero-frontal cirri. Latero-frontal cirri and para-latero-frontal cilia occur on ordinary, principal, and transitional gill filaments. Frontal ciliary tracts of ordinary filaments are divided into a central, ventrally directed coarse tract, flanked on either side by a dorsally directed fine ciliary tract. The coarse tract is covered by cirri which are comprised of five to eight cilia, while the fine frontal tracts are made up of individually functioning cilia. The frontal ciliary tracts of principal and transitional filaments bear only dorsally directed fine cilia. The unique direction of effective beat of the coarse frontal cirri of ordinary filaments, in combination with the action of fine frontal cilia and the strategic location of mucus producing cells, is used to describe a possible mechanism for the sorting of filtered particles.     

Epitheliocystis disease in the striped bass Morone saxatilis from the Chesapeake Bay.

Epitheliocystis disease in the gills of the striped bass Morone saxatilis from Chesapeake Bay was studied using light and electron microscopy. The epitheliocystis infection appeared synchronous in that all capsules on a single host were at the same stage of development. The disease appeared to begin in a single cell on the gill lamella, which gradually enlarged to form a large cyst, encapsulated by a thick cellular capsule of epithelial origin. The epitheliocystis inclusion was filled with cells of the general morphology and size of rickettsiae; however, the infection was atypical for rickettsiae in that the cells had a dense central nucleoid region and they developed within an inclusion separated from the host cytoplasm.     

Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls

Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.     

Parasitism by the protozoan Perkinsus atlanticus favours the development of opportunistic infections

It has been suggested that opportunistic pathogens could contribute to the mortality of Perkinsus atlanticus-infected clams. Examination of Tapes semidecussatus clams from the northern Mediterranean coast of Spain revealed that while 86% of the clams heavily infected with P. atlanticus were co-infected by bacteria and/or viruses, neither non-infected nor lightly P. atlanticus-infected specimens had bacterial or viral infections. The bacteria, which had a Gram-negative cell wall, were always located in the apical pole of gill epithelial cells and enclosed within membranous compartments. Bacteria-containing cells were hypertrophied and showed dysplasia with loss of cilia and microvilli. The viruses shared ultrastructural, morphologic and cytopathic characteristics of a polyomavirus. Viral particles with icosahedral symmetry were found in both the cytoplasm and the nucleus of numerous cell types. Virus-infected cells showed severe alterations, including hypertrophy, reduction of the intracellular compartments and extrusion of the nuclear envelope. Moreover, gill epithelial cells showed disorganization and swelling of the apical region, which affected the ciliary structure. Our findings show that P. atlanticus parasitism favours the development of opportunistic infections which have detrimental effects in this clam population.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

The ciliary basal apparatus is adapted to the structure and mechanics of the epithelium

The basal apparatuses which anchor the gill cilia in Branchiostoma lanceolatum (Pallas) and the actinopharynx cilia in Calliactis parasitica (Couch) are similar in structure. In C. parasitica the pharynx epithelium and the basal apparatuses are flexible. The basal apparatuses, however, bend in only one direction. This mechanism may permit epithelial flexibility whilst maintaining a similar basal orientation between cilia. In B. lanceolatum the ciliated gill epithelia are mechanically stable but the epithelial surfaces are curved. The basal apparatuses may correct for this curvature, with short rootlets between the distal centrioles (basal bodies) and the cell membranes, so that their cilia also share a common orientation. A common basal orientation between cilia is important for their coordination. The degree of coordination depends upon the function of the cilia; water-propelling cilia are more precisely coordinated than mucus-propelling cilia. Much of the structural diversity of ciliary basal apparatuses in Metazoa may be due to variation in the demands of anchoring functionally different cilia to epithelia which have different structural and mechanical properties.     

The major outer membrane protein rOmpB of spotted fever group rickettsiae functions in the rickettsial adherence to and invasion of Vero cells

The role of one of the major outer membrane proteins, rOmpB, of spotted fever group rickettsiae was examined. Antibodies generated against native rOmpB inhibited plaque formation by Rickettsia japonica in Vero cells when applied at the time of inoculation of the rickettsiae. However, antibodies to heat-denatured rOmpB did not. Moreover, the soluble recombinant rOmpB also inhibited plaque formation to some extent. Thus it seems that rOmpB functions at least in the adherence of rickettsiae to host cells. To obtain direct evidence of its function in the adherence to and invasion of Vero cells, we generated Escherichia coli transformed by the vector pET-22b(+) inserted with the ompB open reading frame of R. japonica. The recombinant bacteria expressed a 165-kDa protein consistent with the precursor of rOmpB. The protein reacted with monoclonal antibodies to heat-labile epitopes of rOmpB. Immunofluorescence of the recombinant bacteria demonstrated surface expression of the protein. It was shown by light microscopy and transmission and scanning electron microscopy that the bacteria adhered to and invaded Vero cells. Thus, although the recombinant precursor rOmpB was not processed on the outer membrane of E. coli, it functions during these steps. The manner of entry was similar to that of rickettsiae although at a slower rate.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

Lipid composition of mechanosensitive cell cilia and mechanoreceptor organ of bivalve molluscs

The lipid composition of the mechanoreceptive organ of two marine molluscs in comparison with other molluscan tissues and of isolated cilia of sense organ hair cells in comparison with the gill cilia has been studied. It was shown that the lipid composition of the mechanoreceptive organ differs in a number of features from that of other organs and that the ratios of individual phospholipid classes vary in cilia and intact organs. Ciliary aminophospholipids make up to approximately 89%, whereas choline-containing phospholipids--only 6-8% of the total phospholipid pool. No qualitative phospholipid differences between sense cell cilia and gill cilia were found. However, hair cell cilia contain less phosphatidylethanolamine and choline phospholipids, but more phosphatidylserine and phosphatidylinositol. This suggests that the observed peculiarities of hair cell cilia may play a role in the maintenance of the proper arrangement of hair bundles and the distance between the cilium and stereovilli and/or in the regulation of Ca2(+)-dependent processes.     

Intracellular vesicular trafficking in the gill epithelium of urea-excreting fish

Most teleost fish are ammoniotelic, and relatively few are ureotelic, in which the majority of nitrogenous waste is excreted as urea. This study aimed to determine whether the gill ultrastructure of ureotelic fish might have specific, unique characteristics compared with ammoniotelic fish. The gill morphology was studied in three closely related species of the family Batrachoididae: Opsanus beta, the gulf toadfish; Opsanus tau, the oyster toadfish; and Porichthys notatus, the plainfin midshipman, because prior studies have demonstrated that the two former species are ureotelic and excrete urea in unique, short daily pulses, whereas the latter is ammoniotelic. Ultrastructural studies demonstrated significant trafficking of dense-cored vesicles (50-200 nm) between the Golgi apparatus and the apical membrane of epithelial cells surrounding gill filaments and lamellae in these two Opsanus spp. The material constituting the core of these vesicles was intensely stained by lead salt and was unloaded externally when vesicles contacted the apical membrane. Another characteristic of these urea-secreting fish was the presence of numerous large, black-stained lysosomes, which contained cored vesicles, suggesting a second destination for the dense-cored vesicles. As a working hypothesis, the present data suggest that the urea-transporter protein, recently found in toadfish gills, is inserted in the vesicle. Subsequently, it could serve to either sequester cytosolic urea that ultimately is secreted into the water after contact of these vesicles with the pavement cell apical membrane, or it could allow facilitated diffusion of urea across the plasma membrane following insertion into the membrane. As further comparative evidence, the ammoniotelic P. notatus exhibited neither the vesicular trafficking nor the population of lysosomes both found in Opsanus spp.     

The ultrastructure of the sternal gills forming a striking contrast with the coxal gills in a fresh-water amphipod (Crustacea)

Sternomoera yezoensis has specialized sterna with 21 sternal gills in addition to six pairs of coxal gills. Despite a common high permeability to chloride ions, the cpithelia of these two kinds of gills are diametrically opposed in the polarity of the cell membranemitochondria complex. The coxal gill epithelium (4-6 mum thick) is characterized by a welldeveloped AIS (apical infolding system) associated with a huge number of large mitochondria. The AIS exceeds two-thirds of the epithelial thickness and forms a highly sophisticated, subcuticular labyrinth. On the contrary, the sternal gill epithelium, an extension of the sternal epithelium proper, is extremely thick (10-15 mum) and is characterized by a very deep BIS (basolateral infolding system) associated with numerous slender mitochondria. The BIS reaches nine-tenths of the epithelial thickness and forms a giant, baso-lateral labyrinth. Shallower, less elaborate AIS and BIS without mitochondrial association originate from the opposite sides of these epithelia. Although AIS and BIS interpenetrate in the sternal gill epithelium, they never communicate. The results indicate that in addition to the coxal gills, the sterna with Ihe sternal gills function as transporting as well as respiratory organs, though the functional difference between these two kinds of gills remains to be elucidated.     

Cytological effects of experimental exposure to Hg on the gill epithelium of the European flat oyster Ostrea edulis: ultrastructural and quantitative changes related to bioaccumulation

The significance of ultrastructural changes in the gill epithelial cells as a parameter of detection of Hg exposure in the flat oyster, Ostrea edulis, was tested by a 34-day exposure to 5 microg l(-1) of Hg. The concentration of Hg (38.76 microg g(-1) dry weight) in gill tissue was maximal after 25 days and then decreased. The histological pattern of gill filaments in control samples did not vary throughout the experiment, except for the volume of mucocytes after 4 days of exposure, as an adaptation to experimental conditions. This volume increased significantly and then decreased according to the accumulation of Hg in the gills. After 18 days, absorptive and ciliated cells of the gill epithelium showed blebs in microvilli membranes, discocilia and swollen mitochondrial cristae. Both cell types showed distinct cellular lysis stages after 25 days of exposure. These are the target cells of Hg and other metals and the reported hypertrophy of mucocytes increase occurs in response to pollution by Hg, which could contribute to the detoxification process.     

Ultrastructure of gill cilia and ciliary rootlets of Chaetoderma nitidulum Lovén 1844 (Mollusca, Chaetodermomorpha)

Cilia and associated structures on the gill lamellae on the ctenidum of Chaetoderma nitidulum were studied. The gill cilia are very long and have a whip-like narrow portion distally, where only three microtubule doublets continue to the distal tip. In the transition zone between the cilium and the centriolar triplet section of the basal body there is a dense plate, an aggregation of granules and a ciliary necklace with four strands. Further down there is a short cross-striated basal foot and two conical cross-striated ciliary rootlets. The first rootlet is flattened and directed forward. It connects distally with the basal feet of other adjacent cilia. The second rootlet is rounded in cross-section and vertically directed. The epithelial structures of Chaetoderma show similarities with other Mollusca. We found no structural characters that could support the current hypothesis of a close relationship of Xenoturbella to the Mollusca.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.