Variability of shell repair in the Manila clam Ruditapes philippinarum affected by the Brown Ring Disease: a microstructural and biochemical study

For more than two decades, the Manila clam Ruditapes philippinarum has been regularly affected by Brown Ring Disease (BRD), an epizootic event caused by the bacterium Vibrio tapetis and characterized by the development of a brown deposit on the inner face of valves. Although BRD infection is often lethal, some clams recover by mineralizing a new repair shell layer, which covers the brown deposit and fully isolates it from living tissues. In order to understand this specific shell repair process, the microstructures of repaired zones were compared to those of shells unaffected by BRD. In addition, the organic matrix associated with unaffected shells and to repair patches were extracted and compared by biochemical and immunological techniques. Our results show that the repaired zones exhibit microstructures that resemble the so-called homogeneous microstructure of the internal layer, with some marked differences, like the development of crossed-acicular crystals, which form chevron-like patterns. In the three tested batches of repaired layers, the matrices exhibit certain heterogeneity, i.e., they are partially to widely different from the ones of shells unaffected by BRD, as illustrated by SDS-PAGE and by serological comparisons. Our results strongly suggest a modification of the secretory regime of calcifying mantle cells during the shell repair process. Polyclonal antibodies, which were developed against specific protein fractions of the shell, represent relevant tools for localizing by immunohistology the cells responsible for the repair.     

Impact of Brown Ring Disease on the energy budget of the Manila clam Ruditapes philippinarum

Brown Ring Disease (BRD) is a bacterial disease caused by the pathogen, Vibrio tapetis. The disease induces formation of a brown deposit on inner shell of the Manila clam, Ruditapes philippinarum. Development of this disease is correlated with a decrease in the condition index of infected clams. Experiments were conduced in order to assess the effect of the development of BRD on two parameters affecting the energy balance of the clams: the clearance and the respiration rates. Experiments were performed in a physiological measurement system that allowed simultaneous measures of clearance and respiration rates. During both acclimation and measurements clams were fed with cultured T-iso and temperature was close to seasonal field temperature (10°C). Our results showed that severely diseased clams (conchiolin deposit stage, CDS ≥ 4) are subject to weight loss in comparison to uninfected ones, indicating that BRD induces a disequilibrium in the energy balance. We demonstrated a reduction of the clearance rate of severely diseased clams which led to a decrease in energy acquisition. Respiration rate showed a significant decrease with BRD symptoms, but evidence in the literature allowed us to hypothesize that energy mobilised for an immune response and lesion repair increases overall organism maintenance costs. Both factors should thus contribute to the degradation of the energy balance of diseased clams. Because effects of BRD on naturally infected clams only appears significant for CDS ≥ 4, when brown ring assumes a significant place on the inner shell, we consider that the Manila clam is tolerant of low disease levels.     

Resistance to Brown Ring Disease in the Manila clam, Ruditapes philippinarum: A study of selected stocks showing a recovery process by shell repair

European stocks of the Manila clam Ruditapes philippinarum are affected by the Brown Ring Disease (BRD), which is caused by Vibrio tapetis. BRD is characterized by an accumulation of a brown organic matrix on the inner face of the shell. Clams that recover from BRD develop a white mineralized layer covering the brown matrix. Stocks of clams that showed resistance to BRD development, as enhanced recovery, have been monitored since 2000. We have examined two selected stocks: a Low Susceptibility (LS) stock and a High Susceptibility stock (HS), over three generations. The LS stock showed less evidence of the BRD symptoms, and more evidence of total shell repair, both in the field and following experimental challenge with V. tapetis, indicating that some clams may be less vulnerable to a V. tapetis attack than others. The inner face of the valves of the LS and HS clams of the two last generations were analysed with scanning electron microscopy. Examination of shells from BRD-affected clams showed that during the repair process, calcium crystals were progressively laid down until the affected zone was entirely covered. By the end of the shell repair process, a final organic layer covered the calcium crystal mounds. This layer seemed essential in the recovery process. The results indicate that the shell repair capability of the clams is the principal mechanism implicated in the development of BRD resistance in the Manila clam stocks. However, this resistance did not increase with generation because the broodstock was maintained at a site where selection pressure was low, due to a low prevalence of V. tapetis.     

Shell disease: abnormal conchiolin deposit in the abalone Haliotis tuberculata

Shell disease in the abalone Haliotis tuberculata L. is characterized by a conchiolin deposit on the inner surface of the shell. The gross clinical signs appear similar to the Brown Ring Disease (BRD) of clams. BRD has been extensively described in clams and is known to be responsible for severe mortalities and the collapse of the clam aquaculture industry in western France. In the clam, it was found to be caused by the infection of the mantle by Vibrio tapetis. Brown protein deposits have been observed in various abalone species around the world; some of these have been associated with a fungal infection in New Zealand, but the ones described here are similar to bacterial infections observed in clams. Larger animals appeared to be more affected by the disease, and a positive correlation of the number of successive infections found in the shells with the level of infestation of the shell by borers suggests that boring polychaetes and sponges may be vectors of the disease, or that the parasite infestation may increase the susceptibility of the animal to this infection. There is no evidence, however, that this infection causes mortality in abalone.     

Brown ring disease in clams

Brown Ring Disease (BRD) in the manila clam, Ruditapes philippinarum is a bacterial disease which perturbs the calcification process. This disease occurs in wild and reared clam populations of France, Spain, and occasionally Italy, but has never been reported in Japan from where this species originates. BRD has also been detected in another clam species (Ruditapes decussatus) from France and Spain. Following Koch's classical postulates for pathogen identification, a bacterial strain was identified as the agent responsible for this disease. This bacterium was characterized as belonging to the genus Vibrio and has been termed Vibrio P1 or VP1. The macroscopic sign which characterises this disease is a conchiolin deposit adhering to the inner surface of the shell. To recover from the disease, the clams regenerate their shells by covering the deposit with calcified secretions much like the nacrezation process. Disease and recovery stages have been established for use in epidemiological and experimental studies. Tissue lesions are not systematically observed in diseased clams. Alterations of the digestive gland and the mantle are uniquely detected in the more severe stages of the disease. In all diseased clams, however, the periostracal lamina shows alterations: It is invaded by cell debris and bacteria, and areas of darker melanin-like pigmentation are observed. This disorganized periostracal lamina is not a good substrate for the biomineralization process and, therefore, accumulates forming a deposit on the inner face of the shell. Host-pathogen interactions have been focused at two levels: the internal defense system and the external embedding mechanism. Changes in the hemolymph were observed after challenge with VP1. In the early stages of the disease, an elevation in both circulating hemocytes and peptidase levels appeared; however, a subsequent depression of these parameters was recorded as the disease progressed. An external defense process such as bacterial embedding within periostracal lamina layers is described. Melanization of the periostracal lamina has also been suggested to be an active defense response against bacteria. These new defense processes in Brown Ring Diseased clams could complement the classical hemolymphatic system and, therefore, may act to limit bacterial proliferation within the tissues.     

Isolation of the pathogen Vibrio tapetis and defense parameters in brown ring diseased Manila clams Ruditapes philippinarum cultivated in England

The Manila clam Ruditapes philippinarum was introduced for aquacultural purposes to Europe in the 1970s. In 1987, brown ring disease (BRD), caused by Vibrio tapetis, appeared in clams cultivated in Brou?nou (Finistère, France) and later became increasingly widespread and was reported in cultivated and wild clams existing on the Atlantic coasts of France and Spain. The present study reports, for the first time, the presence of BRD in clams cultivated in England. The etiologic bacterium was isolated and identified using bacteriological and serological techniques. The defence response of affected clams was also studied and significant changes in the hematological and biochemical characteristics of hemolymph and extrapallial fluids were demonstrated. Significant mobilization of hemocytes toward the extrapallial fluids, in contact with the main site of infection (mantle-periostracal lamina area), was observed, suggesting a role for these pseudo-internal compartments in the preservation of clam health.     

Visualization of shell matrix proteins in hemocytes and tissues of the Eastern oyster, Crassostrea virginica

The tissues of the oyster were examined for the presence of shell matrix proteins (SMPs) using a combination of Western, proteomic, and epi-fluorescent microscopy techniques. SMP, including 48 and 55 kDa phosphoproteins, was detected in the epithelial cells of mantle, gill, heart, and adductor muscle and linings of arteries and veins. The 48 kDa SMP circulates continuously within the hemolymph, and is present in the immune system hemocytes. It appears to be secreted from hemocytes on induction of shell repair. We suggest that the 48 and 55 kDa proteins are multifunctional and bridge the process of soft tissue repair and shell formation by mediating cellular activities during immune response as well as interacting with the mineral phase during deposition.     

Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated.     

Effect of temperature on defense parameters in manila clam Ruditapes philippinarum challenged with Vibrio tapetis

Brown Ring Disease (BRD), a vibriosis affecting the clam Ruditapes philippinarum, is present on the Atlantic coasts of Western Europe and is considered to be a cold water disease. The present work investigated the effect of temperature on immune response and its relationships with BRD development. Clams maintained at different temperatures (8, 14 and 21 degrees C) were experimentally challenged with the pathogen Vibrio tapetis, the etiologic agent of BRD. Results demonstrated significant effects of temperature on disease development and on hemolymph immune parameters including total and viable hemocyte counts, lysozyme and leucine aminopeptidase activities. Thirty days after challenge, clams maintained at 21 degrees C displayed significantly higher values for all the measured immune parameters in comparison to specimens incubated at 14 degrees C. Improved performance of the immune system was associated with a low BRD prevalence. The recovery process, which occured mainly at 21 degrees C, was associated with high percentages of viable hemocytes and high activities of leucine amino-peptidase and lysozyme. This laboratory study clearly demonstrates that temperature strongly affects BRD development and clam immune response during infection. Favourable immune status at higher temperature may confer upon the clam a better capacity to fight the disease agent, and therefore to recover more easily.     

Morphology of hemocyte lysis and clotting in the ridgeback prawn,Sicyonia ingentis

Summary Coagulation of hemolymph in the shrimp Sicyonia ingentis was studied using light and electron microscopy. Differential counts of unclotted hemolymph show that 54% of the hemocytes are deposit cells characterized by a high nucleocytoplasmic ratio, a few granules, and cytoplasm filled with distinctive deposits. The remaining hemocytes have numerous large or small granules filling the cytoplasm. Examination of clotted hemolymph to which trypan blue had been added shows that deposit cells lyse, whereas the granulocytes exclude the dye, attach to slides, and extend filopodia. This suggests that deposit cells, not granulocytes, initiate coagulation. Ultrastructural changes in deposit cells were studied at specific times after mixing hemolymph and seawater. Deposit cells fixed immediately after removal from shrimp were shaped like elliptical discs and contained abundant, 50 nm diameter cytoplasmic deposits. After 30 s in seawater, deposit cells displayed several cytoplasmic blebs, and had aggregated the deposits. Cytolysis occurred by 45 s. Linear arrays of deposit appeared to extend through breaks in the plasma membrane, forming filamentous strands that hydrated to produce the clot. At 1 min after withdrawal, spheres of clotted hemolymph were seen, each surrounding a lysed deposit cell. Granulocytes remained relatively unchanged and trapped between adjacent expanding clots. Coagulation via hemocyte lysis is compared with other clotting mechanisms observed in various crustaceans and arthropods.     

First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species,the disk abalone,Haliotis discus discus and the spiny top shell,Turbo cornutus

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes.     

Structure of hematopoietic nodules in the ridgeback prawn,Sicyonia ingentis: Light and electron microscopic observations

The architecture and fine structure of the epigastric hematopoietic nodules of the ridgeback prawn, Sicyonia ingentis, are described. The nodules consist of a highly branched series of tubules that contain the maturing hemocytes within a connective tissue stroma. Hemocytes can exit the hematopoietic nodules by penetrating through fenestrations in the endothelial cell layer into the central hemal space or by migrating through the outer later of capsular cells and associated collagen fibrils. Four hemocyte categories were observed: agranular, small granule with cytoplasmic deposits, small granule without cytoplasmic deposits, and large granule hemocytes. This classification was based upon the presence, size, and type of cytoplasmic granules and the presence of cytoplasmic deposits. Only agranular cells and small granule hemocytes without cytoplasmic deposits appeared capable of division. Intermediate stages were observed between agranular hemocytes and small granule hemocytes with deposits and between small granule hemocytes without deposits and large granule hemocytes, suggesting existence of two distinct hemocyte lines.     

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.     

Repair of molluscan tissue injury: role of PDGF and TGF-beta1

Various cellular and humoral activities of the wound repair process and the effects of PDGF-AB and TGF-beta1 on tissue repair mechanisms in the mollusc Limax maximus are studied by histological and immunocytochemical procedures. Histological examination at different times after the wound production demonstrates that tissue repair is the result of successive and related activities distinguishable by different morphological pictures. In the first hours, an infiltration phase is activated 24 h after the incision, hemocytes stratify at wound margins and actively phagocitize cell debris and damaged tissue in the surrounding area. Moreover, the cells are immunoreactive to anti-IL-1alpha, IL-8 and TNF-alpha antibodies. After 24-72 h, granulation tissue rich in small blood lacunae is formed and the provisional matrix is synthesized and deposited on the base of the new tissue. In histological sections 72 h after injury, the incision is filled with granulation tissue, and at the wound base, a layer of fibrous connective tissue is formed. Hemocytes present in the newly formed blood lacunae and fibroblasts are involved in the synthesis and deposit of extracellular matrix components, i.e. fibronectin, reticular and collagen fibres. Ninety-six h after wound production, the repair process continues and the granulation tissue is more developed. At 192 h, re-epithelialization begins, and this is more evident in the histological sections after 14 days. Hemocytes are immunoreactive to the cytokines at all the times examined. Exogenous administration of PDGF-AB and TGF-beta1 stimulates the tissue healing process through a general acceleration of the activities involved. A larger closing area of clumped hemocytes and a smaller damaged tissue area are observed 24 h after treatment of the wound. At 72 h, the granulation tissue is more developed and more extracellular matrix components are deposited than in the control incision. A larger number of cells express cytokine-like molecules, and re-epithelialization of the wound is accelerated, as 14 days after growth factor treatments almost all the wound area is covered by a layer of cubic epithelial cells, and the alcianophilic cell coat is restored. No differences in the responses of the two growth factors are observed.     

Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.     

染色质乙酰化相关BRD蛋白家族

Bromodomain结构域首先在果蝇蛋白质Brahma中发现,折叠模式独特且高度保守,是最早也是截至目前公认唯一可与乙酰化赖氨酸结合的结构域。BRD蛋白通过结合不同的蛋白质或者定位蛋白质到细胞核发挥精细调节作用。BRD蛋白复合物常特异性识别并结合到染色质组蛋白H3/H4特定的乙酰化赖氨酸残基,从而影响靶基因的转录翻译;该蛋白复合物功能异常通常与多种疾病的发生相关联,表明对转录翻译调节有重要意义。但迄今为止,BRD蛋白复合物修饰染色质机理不明,现有研究提示BRD蛋白复合物维持染色质乙酰化状态,也可以与染色质组蛋白其它位点结合,从整体水平增强组蛋白乙酰化精度和效率。     

The effects of parasitic water mite larvae (Arrenurus spp.) on zygopteran imagoes (Odonata)

Arrenurus larvae, ectoparasitic on zygopteran imagoes, attach to the host's cuticle and tear it to obtain the host's tissue fluids. Within the host's epidermis, each larval mite produces a feeding device, the stylostome, a narrow gelatinous resilient blind sac. Heavy mite infestation brings about several wounds in close proximity, accompanied by loss of more or less extensive areas of the epidermis. Despite wound repair by congregating hemocytes, local lack of epidermis seems to enfeeble the host, presumably owing to desiccation. Heavily mite-loaded zygopterans have lost the typical agility and are easily caught. A mite-induced mortality seems to exist in zygopteran populations; the infestation contributes to reduced longevity. The study of formation and decline of the arrenurid stylostome in zygopterans renders it possible to trace cellular defence reactions under natural conditions. Most stylostomes seem to thwart the ability of the host to recognize them as foreign bodies. The host's defence appears as a two-step reaction: (1) initial hemolymph clotting and deposition of melanin associated with aggregating hemocytes at the penetration site, (2) occasional subsequent melanization and cellular encapsulation of the stylostome.     

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (A_max) and the steroid concentration required for half-maximal induction (EC₅₀) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.     

Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO₃) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.