Variation of the physiological energetics of the bivalve Spisula subtruncata (da Costa, 1778) within an annual cycle

Spisula subtruncata is an infaunal filter-feeding bivalve, which lives in shallow sandy bottoms (2-20 m depth) from Norway to the Atlantic coasts of Morocco, including the Mediterranean Sea. Considering that fisheries of this species have become an important economic resource in some European countries (e.g. The Netherlands), it is of great interest to know the seasonal variation in its physiological energetics. For this purpose, individuals of S. subtruncata were collected and maintained under ambient temperature and seawater conditions of Dutch coastal waters. Physiological processes related to the acquisition and utilisation of energy (e.g. clearance rate [CR], absorption and oxygen uptake) were measured under ambient conditions of the period March 1999 to February 2000. Mean annual clearance and respiration rates (RR) were 0.99 l h⁻¹ and 0.23 ml O₂ h⁻¹ for a standard individual of 250 mg. Values for both clearance and respiration rate were high during spring and summer and low during autumn and winter. Stepwise multiple regression analyses indicated a significant relationship of the clearance rate with temperature and particulate organic matter (POM), whereas respiration rate was significantly related to temperature, absorption rate (AR) of the animals and their reproductive condition. Absorption efficiency (AE) of the food was significantly related to food quality. Scope for growth (SFG) of S. subtruncata, as well as flesh weight of the animals, was high in summer and low in winter.     

An ultrastructural study of connective tissue in mollusc integument III. Cephalopoda

We studied structure and ultrastructure of the subepidermal connective tissue (SEC) of the integument of three cephalopods (Sepia officinalis, Octopus vulgaris and Loligo pealii). In all species, three distinct regions of the SEC were recognised: (a) an outer zone (OZ) that included the dermal-epidermal junction, and consisted of a thin layer of connective tissue containing muscles, (b) an extensive middle zone (MZ) containing a compact network of collagen fibres and numerous cells, (c) an inner zone (IZ) of loose connective tissue that merged with muscular fascia. This arrangement differs from that in bivalves and gastropods and recalls vertebrate integument. The dermal-epidermal junction of cephalopods differed from that of bivalves, gastropods and mammals in that the epidermal cells did not possess hemidesmosomes, and their intermediate filaments terminated directly in the plasmamembrane. The thick (120-500 nm) basal membrane (BM) had a superficial zone containing a regular array of granules; a lamina densa composed of a compact network of small filaments and granules; and an IZ distinguished by expansions of granular material protruding into underlying structures. Collagen fibres contained fibroblast-derived cytoplasmic thread, running through their centres and were surrounded by granular material that joins them to adjacent fibres. The collagen fibrils were of medium diameter (30-80 nm) had the typical ultrastructure of fibrillar collagens, and were surrounded by abundant interfibrillar material. The hypodermis was loose, with a network of small bundles of collagen fibrils. Cephalopod integument appears to represent a major evolutionary step distinguishing this class of molluscs.     

Tissue damage and leukocytic infiltration following attachment of the mite Unionicola intermedia to the gills of the bivalve mollusc Anodonta anatina

Unionicola intermedia lives as a parasite in the gill region of Anodonta anatina. It attaches to the gills of its host by means of the pedipalps which cause displacement, rupture, and erosion of the gill epithelium. The pedipalps are sunk deeply into the underlying connective tissue of the gills and produce a leukocytic infiltration into the damaged area and a consequent edema of the gill filaments.     

An ultrastructural study of connective tissue in mollusc integument: II. Gastropoda

We studied the ultrastructure of the subepidermal connective tissue (SEC) in different zones of the integument in terrestrial, marine and freshwater gastropods (eight species). In all cases, the SEC was a layer of loose connective tissue between the basal membrane (BM) of the epidermis and the connective tissue of the deeper muscle layers. It was of monotonous structure and not differentiated into layers such as are found in mammalian dermis. The extracellular matrix (ECM) consisted of a network of collagen fibrils of variable diameter, with abundant anchoring devices and proteoglycans. In six species, variables quantities of haemocyanin were present within haemocoelic sinuses present in the SEC. The thickness and density of the BM varied from species to species, as well as within species in the various zones of integument. The ultrastructure of the lamina densa (LD) was indistinguishable from that of BM in bivalves and similar to that in mammals, although basotubules and double pegs were absent. An irregularly spaced lamina lucida was usually present and was often shot thorough with filaments and small protrusions of the LD that connected with epithelial plasma membrane or with hemidesmosomes. A lamina fibroreticularis was not present. LD protrusions characterize the connection between BM and the ECM of SEC. In the terrestrial gastropods, a spongy matrix with ultrastructure closely similar to LD occupied large tracts of the SEC. In the mantle region of Arion rufus, the integumental SEC contained large cavities filled with spherical concretions, probably representing rudiments of a shell. In the mantle where the integument contained abundant muscle fibres, the BM was thick and directly connected to the ECM of the SEC which consisted of compact laminae of collagen fibrils with abundant anchoring devices. Along the edge of the foot of Patella ulyssiponensis, the SEC contained a layer of paramyosinic muscle fibres adhering to the epidermis. No differences or gradations in integumental SEC structure could be related to the phylogenetic position of the species examined.     

An ultrastructural study of connective tissue in mollusc integument: I. Bivalvia

The ultrastructure of the subepidermal connective tissue (SEC) in different areas of the integument of the bivalves Callista chione, Pecten jacobaeus, Mytilus galloprovincialis and Ostrea edulis was studied by transmission electron microscopy. The main organisation of the SEC was broadly similar in all species: the SEC was connected to the epidermis by a basement membrane and merged directly with the deeper connective tissue surrounding muscles. The SEC was not differentiated into layers like the papillary and reticular dermis of mammals, however, the architecture, thickness and shape of the basement membrane varied from species to species, as well as within species (in the foot, central or marginal zones of the mantle). The ultrastructure of the lamina densa was broadly similar to that in mammals: although basotubules and double pegs were absent, proteoglycans and rod-like units homologous to 'double tracks' were always abundant. A zone similar to the lamina lucida was irregularly present and was shot thorough with small protrusions of the lamina densa that connected with the epithelial hemidesmosomes or focal adhesions. Nevertheless zones were observed where the lamina densa fuse directly to the epithelial plasmamembrane. This variability of connection may be related to the various types of epidermal cell. A lamina fibroreticularis was not recognized since anchoring fibrils and microfibrils were not present; lamina densa protrusions into the extracellular matrix (ECM) of SEC characterize the connection between basement membrane and SEC. Collagen fibrils were small and of constant diameter and were never organised into fibres. Anchoring devices - similar to the anchoring plaques of mammalian dermis - were abundant and scattered between SEC collagen fibrils. The orange-pink pigmentation of C. chione seems due to electron-dense granules embedded within the connective ECM.     

The first stage larva of brugia pahangi in Aedes togoi: An ultrastructural study

Lehane M. J. 1978. The first stage larva of Brugi pahangi in Aedes togoi: an ultrastructural study. International Journal for Parasitology8: 207–218. The ultrastructure of the first stage larva in the mosquito is described up to the onset of the first cuticular moult. The following structural changes from the microfilarial stage have been noted at this time; the numbers of muscle cells have increased, usually to four with a maximum of six in each intercordai quadrant ; part of the pharyngeal thread has formed into a knot ; the intestinal lumen has developed and is surrounded in any given transverse section by up to four intestinal cells and the inner body has largely been lost; the anal apparatus has enlarged and developed; the excretory apparatus has largely degenerated. The functional morphology of the various organ systems described is discussed.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

An ultrastructural study and stereological analysis of the colon wall in the cockroach Periplaneta americana

The colonic epithelium has been examined for ultrastructural evidence of physiological activity. The cells show extensive folding of the apical plasma membrane, associated with mitochondria and an internal coating of particles about 120 Å diameter. Anteriorly many apical infoldings are dilated at the tip to form substantial extracellular spaces up to 0.8 μm wide. Narrow intercellular channels are present, opening to the haemolymph side of the epithelium. Pinocytosis is seen frequently at the basal surface. The surface densities of apical plasma membranes were not significantly different in the posterior mid-gut and colon. Similarly the volume densities of mitochondria were equal in the colonic epithelium and rectal pads, but the surface density of outer mitochondrial membranes was greater in the colon. It is suggested that the colon may absorb organic solutes from the gut lumen.     

An ultrastructural analysis of the salivary system of the terrestrial mollusc, Limax maximus.

The bilateral salivary glands, ducts, and nerves of the giant garden slug Limax maximus control the secretion of saliva and its transport to the buccal mass. Each salivary nerve, which originates at the buccal ganglion, contains over 3000 axon profiles. The axons innervate the musculature of the duct and branch within the gland. The salivary duct is composed of several muscular layers surrounding an epithelial layer which lines the duct lumen. The morphology of the duct epithelium indicates that it may function in ion or water balance. The salivary gland contains four major types of secretory cells. The secretory products are released from vacuoles in the gland cells, and are presumably transported by cilia in the collecting ducts of the gland into the larger muscular ducts.     

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.     

Hemolymph cells of the bivalve mollusc Mercenaria mercenaria: an electron microscopical study.

The hemolymph cells of Mercenaria mercenaria were studied with the transmission electron microscope. Three morphological types of cells, granulocytes, hyalinocytes, and fibrocytes, are distinguishable and their fine structural characteristics are described. However, as a result of analyzing the fine structural features of the so-called fibrocytes of M. mercenaria, i.e., the inclusion of large aggregates of glycogen granules in their cytoplasm and the occurrence of primary phagosomes enclosing partially degraded exogenous material and digestive lamellae, it is suggested that fibrocytes are actually granulocytes which are at the terminal phase of their physiologic cycle relative to the degradation of phagocytized nonself materials. The cytoplasmic granules of M. mercenaria granulocytes are structurally different from those of Crassostrea virginica in that they are delimited by a unit membrane, rather than by a complex wall, and include a homogenously electron-dense material. Lipidlike droplets are reported from both granulocytes and hyalinocytes of M. mercenaria for the first time.     

Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

A histological and ultrastructural study of the cells of the mantle edge of a marine bivalve, Cerastoderma edule

The cells of the mantle edge of Cerastoderma edule are described after light and electron microscopical observations. Histochemical tests for calcium in the mantle edge and digestive gland (Dahl, 1952; McGee-Russell, 1958) and analytical electron microscopy of the mantle edge of C. edule both failed to show calcium. Similar results were obtained for Mytilus edulis and Chlamys opercularis. However, calcium was detected in the digestive gland of the terrestrial gastropod Helix aspersa. The outer secretory fold of the mantle edge is composed of tall columnar cells. These cells have highly convoluted lateral cell membranes with which many mitochondria are closely associated. These features are indicative of an ion pump which could move calcium from the mantle space to the extrapallial cavity (compare with Bubel's findings, 1973b). There are many features of the cells lining the periostracal groove of C. edule that have not been reported previously (e.g. Bubel, 1973b) and which are now discussed. The periostracal sheet arises within a line of basal cells in the fundus of the periostracal groove. Within these cells the periostracum in section has a spiral form. It is suggested that the newly formed periostracum adheres to the microvillous border through secretions produced from the middle fold cells lining the groove. During its passage along the groove the periostracum is gradually thickened by secretions from the outer fold cells.     

Recycling of mast cells following degranulation in vitro: An ultrastructural study

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting cells or after degranulation by exposure to compound $\text{48}\text{80}$, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of ‘recycling’. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. It may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.     

Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus.

The proliferative disease systemic neoplasia, also termed hemic neoplasia or disseminated sarcoma, was studied in four Puget Sound, Washington populations of the bay mussel (Mytilus sp.). Using flow cytometric measurement of DAPI-stained cells withdrawn from the hemolymph, DNA content frequency histograms were generated for 73 individuals affected by the disease. The cells manifesting systemic neoplasia were found to exist as either of two separate types, characterized by G0G1 phase nuclear DNA contents of either approximately 4.9 x haploid (pentaploid form) or approximately 3.8 x haploid (tetraploid form). The two disease forms were found to coexist in all four mussel populations sampled, with overall relative prevalences of 66% pentaploid form, 29% tetraploid form, and 5% exhibiting both disease forms simultaneously. These findings represent the first unequivocal demonstration of multiple cell types in a bivalve neoplasia. The two forms appear to represent separate pathogenetic processes rather than sequential stages of a single pathogenesis. Two cell cycling parameters associated with proliferative activity were employed to compare the alternate forms: (i) the percentage of cells assigned to the DNA Synthesis (S) phase of the neoplastic cell cycle, and (ii) the proportion of neoplastic cell mitotic figures in hemocytological preparations. Mean values for both parameters were significantly higher for mussels with the tetraploid form of the disease, suggesting a higher rate of proliferation relative to the pentaploid form. Qualitatively, cells of the tetraploid form contained slightly lower nuclear and cytoplasmic volumes compared to those of the pentaploid form. An observed wide variation in neoplastic cell nuclear size within either disease form may reflect the distribution of cells in the G0G1, S, and G2M phases of the cell cycle. Potential etiologic relationships between the two forms are discussed.     

An ultrastructural investigation of the surface microbiota present on the leaves and reproductive structures of the resurrection plant Myrothamnus flabellifolia

The leaves, flower and stems of the southern African angiosperm resurrection plant Myrothamnus flabellifolia were investigated at the ultrastructural level to determine the source of previously reported fungal contamination. Fungal mycelia and hyphae of the genera Aspergillus and Penicillium were found localized to the hydathodes of the leaves and stigmatic surfaces of the female flowers in both desiccated and hydrated specimens. A waxy bacterium of the genus Bacillus was found to colonise the waxy epidermal surfaces of the leaves and flowers which was also where fungal cells were found to be absent. It is suggested that the wax like deposits within the leaves and stems as well as over the epidermal surface prevent the growth of the fungal organisms. These fungi opportunistically invade moist surfaces, such as the floral stigmas, during periods of moisture availability and may thus negatively impact plant development.     

41 Incidence of paralytic shellfish toxin in bivalve mollusc tissue from the oregon coast

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin‐producing organisms is unknown.