Gill structure in Lucina pectinata (Bivalvia: Lucinidae) with reference to hemoglobin in bivalves with symbiotic sulphur-oxidizing bacteria

Lucina pectinata is a large tropical Lucinidae which is characterized by abundant tissue hemoglobin in its deep-red gills. In the present paper, hemoglobin is described as being located in cytoplasmic dark patches of the bacteriocytes together with a cystine-rich protein. Large microbodies contain a non-hemoglobin heme-compound which is identified with a previously described non-protein-bound hematin; however, it has not been established whether this heme is involved in a sulphur-oxidizing system or represents a catabolic by-product of hemoglobin. Electron-lucent vesicles are associated with the basal microbodies but their function is, so far, unknown. In addition, the bacteriocytes have been observed to have direct contact with sea water, modulated by large intercalary cells which overlap the bacteriocytes on their margin. Such relationships between bacteriocytes and intercalary cells, as well as their cytological features, are different from those observed in lucinid species inhabiting sea-grass beds, but very similar to those observed in Calyptogena magnifica. From the congruence between the shallow-water Lucinidae L. pectinata, inhabiting mangrove swamps, and the deep-sea Vesicomyidae C. magnifica, found at hydrothermal vents, we conclude that such features are likely to be adaptative to high-sulphide environments, notwithstanding the phylogenetic distance.     

Sulfide-oxidizing bacteria in the burrowing echinoid,Echinocardium cordatum (Echinodermata)

Symbiotic filamentous bacteria thrive in the intestinal caecum of the deposit-feeding echinoid Echinocardium cordatum. Specimens of E. cordatum were collected at Wimereux (Nord Pas-de-Calais, France) in 1991. Their symbiotic bacteria build nodules by forming multilayered mats around detrital particles that enter the caecum. The morphological features of the bacteria are those of Thiothrix, a sulfide-oxidizing genus. The filaments, which may form rosettes, are sheathed and made by a succession of hundreds of rod-shaped bacteria which store elemental sulfur in the presence of external sulfide. Live bacteria are restricted to the outer layers of the nodules. Their sulfide-oxidizing activity was investigated, using a Biological Oxygen Monitor, by measuring the O₂-consumption when reduced sulfur compounds are provided. They oxidize thiosulfate and sulfide. Optimal sulfide oxidation occurs at intermediary pO₂ (100 to 160 M O₂l^-1). Spectrophotometry has confirmed that the sulfur content of the filamentous symbiotic sulfideoxidizing bacteria depends on the presence of external sulfide. This is the first report of symbiotic intradigestive Thiothrixspp.-like bacteria; it lengthens the list of symbioses between sulfide-oxidizing bacteria and invertebrates from sulfide-rich habitats.     

Methanotrophic symbiont location and fate of carbon incorporated from methane in a hydrocarbon seep mussel

Seep Mytilid Ia (SMIa), an undescribed mussel found at hydrocarbon seeps in the Gulf of Mexico, harbors intracellular methanotrophic symbionts. Two techniques were used to address the hypothesis that host digestion of symbionts is a significant mechanism of carbon transfer from symbiont to host in the SMIa association: lysosomal enzyme cytochemistry and ¹⁴C tissue autoradiography. Acid phosphatase activity was consistently localized in the Golgi apparatus and associated vesicles of gill cells, but was detected around bacteria in only three of approximately 50 bacteriocytes examined. These results indicate that the cellular equipment necessary for lysosomal digestion of symbionts is present in host bacteriocytes, but that acid phosphatase activity in symbiont vacuoles is rare at a given point in time. Tissue autoradiography was conducted with mussels collected in September 1992 to document carbon fixation by symbionts and follow the time course of transfer to host tissues. No asymbiotic host cell type showed a significant increase in relative grain density until at least 1 d after the end of incubation with ¹⁴C-methane. The ratio of label in the basal portion of bacteriocytes to total bacteriocyte label did not show a significant increase until 10 d after the end of the incubation period, indicating a slow increase of labeled carbon in the putative residual bodies, containing the remnants of lysosomal digestion. These results are consistent with the hypothesis that host digestion of symbionts is one route of nutrient acquisition in SMIa. Intracellular methanotrophic bacteria were found outside of the gill in SMIa juveniles, in mantle and foot epithelial tissues previously believed to be symbiont-free. These extra-gill symbionts and their host cells are morphologically similar to their gill counterparts and, like the gill symbionts, actively fix carbon from methane. Received: 29 March 1997 / Accepted: 12 May 1997     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

In situ characterization of sulphur in gill-endosymbionts of the shallow water lucinid Codakia orbicularis (Linné, 1758) by high-pressure cryofixation and EFTEM microanalysis

In this study, Codakia orbicularis gill-tissues were cryo-fixed by using high-pressure freezing, a freeze substitution process and finally by cryo-embedding in Lowicryl. Ultrathin sections were then used for an EFTEM microanalysis. Results show that intracellular bacterial symbionts contain elemental sulphur in periplasmic vesicles as indicated by conventional TEM. When sulphur is temporarily depleted in the environment, such structures may act as energy sources for bacterial metabolism. Moreover, sulphate was detected in the cytoplasm of the bacterial symbionts, suggesting the oxidation of elemental sulphur, located in periplasmic granules, to sulphate (the final step in sulphur oxidation) by these chemoautotrophic bacteria. To assess the effects of host starvation on the bacterial sulphur content, adult individuals of C. orbicularis were maintained in starvation for 6 weeks in sterile artificial seawater depleted in sulphur. During starvation, both (1) the number of bacteria inside the bacteriocytes and (2) the number of periplasmic granules per prokaryotic cell decreased. The content of the remaining periplasmic granules had been modified to sulphate. This observation suggests that bacterial gill-endosymbionts used the elemental sulphur in their periplasmic granules as stored substrate for oxidation in order to produce energy in case of sulphur depletion.     

Histochemical and ultrastructural characterisation of mantle storage cells in the hydrothermal-vent bivalve Bathymodiolus azoricus

This study reports on two types of storage cells that are present in the mantle connective tissue of the deep-sea mussel Bathymodiolus azoricus. One type corresponds to the adipogranular cells, a kind of storage cell previously described in other bivalves. In these cells extensive regions of the cytoplasm are filled with glycogen deposits and these zones became strongly stained after histochemical (PAS) or ultrastructural detection of polysaccharides. Several lipid droplets and membrane bound granules containing homogeneous electron-dense material are also present in adipogranular cells. A second type of cell contains large lysosomes in addition to numerous lipid droplets, but lacking cytoplasmic glycogen deposits. Due to these characteristics we named them adipolysosomal cells. They can be identified in semi-thin sections stained with PAS reaction because the lysosomes are the only positively stained structures. In the connective tissue of the mantle, some cells containing many lysosomes and a few lipid droplets were also observed. These cells differ from the adipolysosomal cells mainly because they have a reduced amount of lipid reserves, and could be an initial stage in the development of adipolysosomal cells. The vesicular connective tissue cells that in other Mytilidae are specialised in glycogen storage were not detected in B. azoricus. The reserves accumulated in the two types of storage cells described in B. azoricus may be important for the survival of these hydrothermal-vent bivalves if their nutrition is affected by a temporary loss or reduction of endosymbiotic bacteria due to sulphide and/or methane shortage caused by oscillations in vent activity.     

Ultrastructure of the gill of the hydrothermal vent bivalveCalyptogena magnifica,with a discussion of its nutrition

Calyptogena magnifica Boss and Turner, 1980, a new Vesicomyidae found during the Galápagos expedition in hydrothermal vents of the East Pacific Rise, was collected in the same Rise at 21°N during the Oasis expedition (March 1982), and samples of the gill were fixed for ultrastructural observations. The large size and structure of the gill indicate that this is the organ mainly involved in the nutritional processes ofC. magnifica. Despite the classic structural appearance of the external cilia of its gill, and an obvious production of mucus,C. magnifica is not a filter-feeder, as it does not use filtering processes to provide its major source of nutrition. Negligible particulate transfer is evidenced by reduction of the ciliary groove, of the labial palps and of the digestive tube, as well as by the absence of mucous strings. Histological and ultrastructural observations endorse the hypothesis that endocellular chemoautotrophic bacteria play an important role in the nutrition of the clam. Except for a superficial zone of ciliated cells, most of the gill tissue is comprised of cells which appear to be bacteriocytes, and which are perfectly integrated into the gill tissue and contain abundant and normally reproducing bacteria. The differences observed in the structure of the bacteriocytes suggest a cyclic process of their colonization by bacteria, their possible resorption, and their replacement by new bacteria-infected cells. Energetic substrates (sulfides and organic molecules) are probably directly absorbed by the bacteriocytes through the microvilli of the epithelial cells. Abundant fingerprint-like mitochondria in ciliate cells attest to a particularly high metabolic activity, perhaps related to active biosynthesis.     

Phenotypic variations in the gills of the symbiont-containing bivalve Lucinoma aequizonata

The marine bivalve Lucinoma aequizonata (Lucinidae) maintains a population of sulfide-oxidizing chemoautotrophic bacteria in its gill tissue. These are housed in large numbers intracellularly in specialized host cells, termed bacteriocytes. In a natural population of L. aequizonata, striking variations of the gill colors occur, ranging from yellow to grey, brown and black. The aim of the present study was to investigate how this phenomenon relates to the physiology and numbers of the symbiont population. Our results show that in aquarium-maintained animals, black gills contained fewer numbers of bacteria as well as lower concentrations of sulfur and total protein. Nitrate respiration was stimulated by sulfide (but not by thiosulfate) 33-fold in homogenates of black gills and threefold in yellow gill homogenates. The total rates of sulfide-stimulated nitrate respiration were the same. Oxygen respiration could be measured in animals with yellow gills but not in animals with black gills. The cumulative data suggest that black-gilled clams maintained in the aquarium represent a starvation state. When collected from their natural habitat black gills contain the same number of bacteria as yellow gills. Also, no significant difference in glycogen concentrations of the host tissues was observed. Therefore, starvation is unlikely the cause of black gill color in a natural population. Alternative sources of nutrition to sulfur-based metabolism are discussed. Denaturing gradient gel electrophoresis (DGGE) performed on the different gill tissues, as well as on isolated symbionts, resulted in a single gill symbiont amplification product, the sequence of which is identical to published data. These findings provide molecular evidence that one dominant phylotype is present in the morphologically different gill tissues. Nevertheless, the presence of other phylotypes cannot formally be excluded. The implications of this study are that the gill of L. aequizonata is a highly dynamic organ which lends itself to more detailed studies regarding the molecular and cellular processes underlying nutrient transfer, regulation of bacterial numbers and host–symbiont communication. Received: 1 September 1999 / Accepted: 1 February 2000     

Tissue and sub-cellular distribution of Fe,Cu, Zn and 210Po in the abalone Haliotis rubra

The tissue and sub-cellular distribution of Fe, Cu, Zn and the naturally occurring radionuclide polonium-210 was determined in the gastropod mollusc Haliotis rubra collected from Western Port Bay, Australia, between March and July 1988. The highest concentrations of the metals, with the exception of Cu, were found in the digestive gland. Copper was more uniformly distributed, with tissues that are more vasculated having higher concentrations. Ultrastructural examination of the digestive gland, gill and kidney showed dense membrane-bound granules within the cytoplasm. Elemental analysis of the granules by electron probe x-ray microanalysis indicated that the granules in the digestive gland and gill contained high concentrations of iron, with small amounts of copper and zinc. In contrast, the metal-containing granules in the kidney were predominantly composed of iron, copper and phosphorus, with variable contributions of sodium potassium, and calcium. Homogenisation and fractionation of the digestive gland by differential centrifugation confirmed that approximately 80, 10, 90 and 50% of the total homogenate Fe, Cu, Zn and ²¹⁰Po, respectively, sedimented at 1200xg. In the haemolymph, all the elements studied were associated with the soluble high molecular weight component of the serum, not with the amoebocytes. ²¹⁰Po was present in the mucus-secreting hypobranchial glands at about half the concentration found in gill tissue.     

Comparison of proton-specific ATPase activities in plume and root tissues of two co-occurring hydrocarbon seep tubeworm species Lamellibrachia luymesi and Seepiophila jonesi

Lamellibrachia luymesi and Seepiophila jonesi are co-occurring species of vestimentiferan tubeworms found at hydrocarbon seepage sites on the upper Louisiana slope of the Gulf of Mexico. Like all vestimentiferans, they rely on internal sulfide-oxidizing symbiotic bacteria for nutrition. These symbionts produce hydrogen ions as a byproduct of sulfide oxidation, which the host tubeworm needs to eliminate to prevent acidosis. The hydrothermal vent tubeworm Riftia pachyptila uses a high activity of P- and V-type H⁺-ATPases located in its plume epithelium to excrete protons. Unlike R. pachyptila, the seep species grow a posterior root, which they can use in addition to their plumes as a nutrient exchange surface. In this study we measured the ATPase activities of plume and root tissues collected from L. luymesi and S. jonesi, and used a combination of inhibitors to determine the relative activities of P- and V-type H⁺-ATPases. We found that the total H⁺-ATPase activity of their plumes was approximately 14 μmol h⁻¹ g⁻¹ wet weight, and that of their roots was between 5 and 7 μmol h⁻¹ g⁻¹ wet weight. These activities were more than ten times lower than those measured in R. pachyptila. We suggest that seep tubeworms might use passive channels to eliminate protons across their roots, in addition to ATP-dependant proton pumps located in their plumes and roots. In addition, we found strong differences between the types of ATPase activities in the plumes of L. luymesi and S. jonesi. While the H⁺-ATPase activity of L. luymesi plumes is dominated by P-type ATPases, S. jonesi has an unusually high activity of V-type H⁺-ATPases. We suggest that S. jonesi relies on its high V-type H⁺-ATPase activity to drive carbon dioxide uptake across its plume surface. L. luymesi, on the other hand, might rely partially on bicarbonate uptake across its root.     

Single and combined effects of heavy metals and hormones on lysosomes of haemolymph cells from the mussel Mytilus galloprovincialis

Effects of heavy metals on lysosomes were studied in living cells from the mussel (Mytilus galloprovincialis Lam.). Haemolymph cells were obtained from the mussel adductor muscle, stained with neutral red (NR), and analysed by digital imaging to evaluate NR retention times within lysosomes. Exposure to Hg²⁺, Cd²⁺ and Cu²⁺ induced a reduction of NR retention time, indicating lysosomal membrane destabilisation. The intensity of these effects was correlated with the metal affinity for sulfhydryls. In contrast, Zn²⁺ showed no effect on lysosomes. Moreover, 200 μM Zn²⁺ protected lysosomes against the effects of Cd²⁺ and Cu²⁺, but not against Hg²⁺. Cell loading with the fluorescent pH probe Lyso Sensor followed by digital imaging showed a rise of lysosomal pH induced by Cd²⁺ and Hg²⁺, while Zn²⁺ prevented the effect of Cd²⁺ and also partially that of Hg²⁺. The different protective effect of Zn²⁺ against Hg²⁺ suggests a dual action of Hg²⁺ on lysosomes, possibly involving both membrane destabilisation and proton pump inhibition. Cell exposure to 17 β-estradiol also caused a reduction of NR retention time, which was synergistic to that of Hg²⁺. This suggests a common pathway between metals and hormone, possibly involving Ca²⁺ signaling. Received: 17 November 1999 / Accepted: 29 June 2000     

Cellular and ultrastructural changes in the endoderm of the temperate sea anemone Anemonia viridis as a result of increased temperature

Exposure of the temperate sea anemone Anemonia viridis Forskål to increased seawater temperature (from 16 to 26°C) reduced the lysosomal latency of coelenterate tissues. Lysosomes in the mesenterial filaments of anemones were destabilised by increased temperature, with greater destabilisation in heat-shocked symbiotic anemones than in heat-shocked aposymbiotic anemones in the early stages of the experiment. Lysosomal enzyme activity in zooxanthellae from heat-shocked symbiotic anemones was associated with the algal membranes and the cytoplasm of degenerate algal cells. While the relationship between host coelenterate and symbiotic alga may confer many benefits under normal conditions, comparison of the responses of symbiotic and aposymbiotic anemones to heat shock suggests that there may be disadvantages for symbiotic anemones under stress.     

An electron-microscope study of periostracum formation in some marine bivalves. II. The cells lining the periostracal groove

The histology and ultrastructure of the epithelial cells of the outer and middle mantle folds of the bivalves Mytilus edulis (L), Cardium edule (L), Macoma balthica (L) and Nucula sulcata Bronn are described. The cells lining the inner face of the outer fold exhibit a prominent granular endoplasmic reticulum, and Golgi complexes which are concerned with the elaboration of granules and vesicles eventually incorporated into the periostracum. A gradual reduction in the protein synthetic apparatus occurs towards the tip of the fold. Within the cells, it is proposed that the ovoid inclusion bodies are lysosomes and that they control the rate of secretion. The cells of the middle fold are cuboidal in appearance. Those of M. edulis and N. sulcata exhibit prominent granules, whereas those of C. edule and M. balthica possess vesicles. The cells of M. edulis differ from the others in possessing stout bundles of filaments, which occupy large areas of the cell and constitute a cell web. The cells of the epithelium in all cases do not appear to be implicated in periostracum formation.     

Molecular response of the sponge Suberites domuncula to bacterial infection

The aim of this study was the documentation of the molecular immune response of Suberites domuncula upon bacterial infection. Additionally, the bacteria that are naturally present in the sponge after prolonged aquarium maintenance were characterized. After 6 months of maintenance of S. domuncula in seawater aquaria, only one bacterial 16S rDNA sequence could be recovered, which belongs to the genus Pseudomonas. Concomitantly, morphologically uniform bacteria were found encapsulated in bacteriocytes. These findings indicate that certain bacteria, possibly of the genus Pseudomonas, are able to persist for long periods in host bacteriocytes. Subsequent to performing a previously established infection assay with S. domuncula, a potentially pathogenic Vibrio sp. was isolated from the tissues. Furthermore, the host tissue disintegrated and asexual propagation bodies (gemmules) were formed. In order to gain insights into the molecular events occurring after bacterial infection, the stress-response kinases, p38 protein kinase and JNK protein kinase, were analyzed. It is demonstrated that these two kinases are activated (phosphorylated) upon incubation of the tissue with the bacterial endotoxin lipopolysaccharide (LPS). Moreover, LPS strongly inhibits protein synthesis. It is concluded that there are many functionally different interactions between S. domuncula and bacteria and that the animal possesses mechanisms to differentiate between bacteria and to respond accordingly.     

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S⁼ and stimulated by S₂O₃ ⁼. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO₂-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other thiobiotic animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.     

Inception of formation and early morphogenesis of the bacterial light organ of the sea urchin cardinalfish, <Emphasis Type="Italic">Siphamia versicolor</Emphasis>

The cardinalfish Siphamia versicolor (Perciformes: Apogonidae) forms a bioluminescent symbiosis with the marine luminous bacterium Photobacterium mandapamensis, harboring the bacteria in a ventral, disc-shaped light organ and using the bacterial light apparently for counterillumination and attracting prey. Little definitive information has been available on the developmental and microbiological events surrounding the initiation of symbiosis, a critical stage in the life history of the fish, in S. versicolor or any of the many other species of bacterially luminous fish. To identify the stage at which light organ formation begins, to determine the origin of cells forming the light organ, and to characterize its bacterial colonization status during development, early developmental stages of S. versicolor obtained and reared from wild-caught mouth-brooding males were examined with histological and microbiological methods. A light organ primordium was not evident in embryos, post-embryos, or pre-release larvae, whereas the light organ began to form within 1 day of release of full-term pre-flexion larvae from the mouths of male fish. Analysis of post-release larvae revealed that the light organ arises from a proliferation and differentiation of intestinal epithelial cells, and that it quickly develops structural complexity, including the formation of chambers and gaps contiguous with the intestinal epithelium. However, the nascent light organ remained uncolonized by the symbiotic bacteria through several days of post-release development, even in the presence of high numbers of the symbiotic bacteria. These results demonstrate that the inception of light organ formation in S. versicolor occurs independently of its symbiotic bacteria and that receptivity to bacterial colonization apparently requires substantial post-release development of the light organ. Larvae therefore most likely acquire their symbiotic bacteria from seawater, during or shortly after the transition from the pre-flexion to the flexion developmental stage.     

Further 237Pu experiments with the plaice Pleuronectes platessa: Subcellular distribution of plutonium in the liver

The subcellular distribution of ²³⁷Pu in the livers of the plaice Pleuronectes platessa has been studied by differential and isopycnic gradient centrifugation. It has been shown that the ²³⁷Pu is particularly accumulated in pellets sedimenting out at 3000 and 6000 g which, by deduction from marker enzyme tests, contain a mixture of mitochondria and lysosomes. Separation of lysosomes from mitochondria was achieved by using a non-lytic detergent injected into the fish 3 d prior to removal of the livers; it was found that ²³⁷Pu was associated with the lysosomal fraction. In this respect, the metabolism of plutonium of the plaice resembles that of the rat.     

Ultrastructural and cytochemical study on interactions between nutrient storage cells and gametogenesis in the mussel Mytilus edulis

The ultrastructural morphology of storage-cell breakdown and replenishment in Mytilus edulis is described for mussels collected at monthly intervals over a period of one year (September 1981 to October 1982) from a site in Cornwall, England. In addition cytochemical staining reactions at light and electron microscope levels are described for the storage cells and the Sertoli cells. The breakdown of adipogranular (ADG) cell components is believed to be a lysosomally-mediated process of controlled autolysis involving fusion of primary lysosomes with the protein granules. Increasing activity for lysosomal enzymes during ADG cell breakdown is demonstrated using quantitative cytochemistry. The presence of glycogen in vesicular connective tissue (VCT) cells is demonstrated using freeze-dried, formaldehyde-vapour-fixed tissue. The breakdown of VCT cells involves sequestration of glycogen from the central reserve into small cytoplasmic vesicles; subsequent release appears to be mediated by eccrine secretion. The presence of arylsulphatase activity in lysosomes within the peripheral cytoplasm of the VCT cells during the winter months indicates that glycogen breakdown may also be lysosomally mediated. The morphology of the Sertoli cells is described; during the early stages of spermatogenesis they are characterized by the presence of numerous lipid droplets which are depleted as development proceeds. Contact between Sertoli cells and developing spermatogonia is maintained by means of long septate junctions. Following spawning, the well developed lysosomal system is evident in the Sertoli cells and appears to be engaged in intracellular digestion of phagocytosed waste sperm and residual cytoplasm. Resorption of the products of gamete degeneration by the gonoduct epithelial cells is described. During regeneration, VCT cells appear to endocytose material; the ADG cells demonstrate extensive arrays of rough endoplasmic reticulum and are occasionally seen undergoing mitosis. A process involving loss of glycogen vesicles from the mantle epithelial cells by means of apocrine sections is described.     

Seasonal cycle of lysosomal enzyme activities in the mantle tissue and isolated cells from the musselMytilus edulis

InMytilus edulis L., gametogenesis takes place in the mantle at the expense of the connective storage tissue. There are two main types of storage cells: vesicular (VC) cells storing large amounts of glycogen and adipogranular (ADG) cells containing large numbers of protein granules, lipid droplets and lesser amounts of glycogen. One of the ways in which stored reserves can be mobilized for gamete formation is by controlled autophagy, in which the cellular constituents are degraded by lysosomes. Mussels were collected from the Menai Strait, North Wales, and monthly measurements made, over two years (1984–1986), of the activities of lysosomal acid hydrolases (acid phosphatase,-N-acetyl-D-glucosaminidase and-glucuronidase) and Cathepsins B and L in the mantle tissue, isolated ADG cells, low-density cells and, during spawning, in the mature oocytes of female mussels. The lysosomal proteinases, Cathepsins B, L and H, were further characterised by activation with thiol compounds and inhibition with thiol blockers and by leupeptin. Because of the low activity in the mantle tissue ofM. edulis, Cathepsin H was not assayed on a seasonal basis. There was a general increase in lysosomal enzyme activity during the winter, which can be related to increased autolysis in the storage cells and to the process of maturation in the developing oocytes. The activity of Cathepsin B was highest in the ADG and low-density cells, implying an important role in proteolysis within the ADG cells. By contrast, Cathepsin L displayed the highest activity in the mature oocytes, suggesting a major function of Cathepsin L in the development and maturation of the oocytes. Two different-glucosidase activities were measured in the monthly assays, one with a pH optimum of 4.5 (acid) and the other at pH 7.5 (neutral). Highest activities of the acid-glycosidase were found in the low-density cells, but there were no significant seasonal changes in the mantle tissue as a whole. Activities of the neutral-glucosidase were low in the ADG cells and mature oocytes, but showed high activities in the mantle tissue, with marked seasonal changes that corresponded to the mobilization of glycogen reserves in the VC cells.     

Temporal variation of lysosomal capacities in relation to susceptibility of mussels, Mytilus edulis, to summer mortality

We examined the temporal variation of lysosomal enzyme activities and lysosomal membrane stability in two stocks of blue mussels (Mytilus edulis L., 1758) from the Magdalen Islands which differ in their susceptibility to summer mass-mortality. The activity of lysosomal enzymes reflects the autolytic capacity which could be used during the transfer of reserves between storage and reproductive tissues or for mobilisation of reserves for energy requirements. The peak of lysosomal enzyme activities in mantle tissue in June 1992 probably was related with autolysis of storage cells to support maturation of the developing gametes. A second peak of lysosomal enzyme activity which occurred in mid- August of 1992 and 1993 may help to cover energetic requirements of maintenance metabolism during this period of high temperatures and reduced food quality. Measures of the destabilization of lysosomal membranes in the digestive gland confirmed that mussels showed a significant stress response in mid-August. Mussels from the stock with a higher susceptibility to summer mortality underwent a longer period of stress and accumulated less glycogen between spawning and the stressful period in mid-August than mussels from the more resistant stock. In conclusion, our field observations demonstrate high activities of lysosomes not only in spawning periods but also during stressful periods of high temperatures and reduced food quality. Received: 23 September 1997 / Accepted: 11 June 1998