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.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Pathology of acute poisoning with the insecticide Sevin in the bent-nosed clam, Macoma nasuta

Toxicity tests of 96-hr duration of the insecticide Sevin were done with adult bent-nosed clams, Macoma nasuta. Sevin concentrations of 15, 20, 25, and 30 mg/liter were used in duplicate tests. The criterion of “death” was the inability of clams to retract siphons or to close valves. About half of the animals so affected were removed from the test solutions and returned to clean sea water to observe if recovery occurred, and others were preserved for histological examination.No “dead” clams recovered within 96 hr after return to clean water. The histopathology consisted primarily of necrosis of epithelial tissue of the gill, mantle, siphon, and suprabranchial gland, and the severity of damage was directly related to the test concentrations. Vacuolization, rupture, and pycnosis of cells occurred. The gills were the most severely affected organs. Epithelial cells of the gill filaments bearing the frontal, laterofrontal, and lateral cilia were sloughed as early as 24 hr after the beginning of exposure to Sevin. About 50% of the exposed clams had lost one or both siphons and also the epithelia on still attached segments within 96 hr of exposure. There were no deaths of control clams, and their tissues were normal.     

Molecular cloning and characterization of SOCS-2 from Manila clam Ruditapes philippinarum

Suppressor of cytokine signaling (SOCS) family members are key regulators of immunological homeostasis. In this study, we have discovered the SOCS-2 member from Manila clam Ruditapes philippinarum and further analyzed its immune responses against lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). Amino acid sequence of RpSOCS-2 consists of cytokine inducible SRC homology 2 (SH2) and SOCS box domains similar to vertebrate SOCS counterparts. It has the highest amino acid identity (41%) with Pacific oyster (Crassostrea gigas) SOCS-2 and showed close evolutional relationship with disk abalone (Haliotis discus discus) SOCS-2. Tissue specific expression results showed that RpSOCS-2 was constitutively expressed in all examined tissues with the highest level in gill tissue of un-challenged clams. RpSOCS-2 mRNA expression was up-regulated by LPS and poly I:C challenge in gills. Discovery of RpSOCS-2 homologue and expression analysis would support for understanding evolutional relationships and their role in innate immune responses in mollusks, respectively.     

Brown muscle disease: Impact on Manila clam Venerupis (=Ruditapes) philippinarum biology

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared.Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD.In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production.Most of the studied parameters underlined a cause–effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression.     

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.     

Molecular Identification of the Ichthyosporean Protist “Pseudoperkinsus tapetis” from the Mytilid Mussel Adipicola pacifica Associated with Submerged Whale Carcasses in Japan

A protist tentatively designated “Pseudoperkinsus tapetis” belonging to the eukaryotic group Ichthyosporea (Mesomycetozoa) was previously isolated from carpet shell clams in Galicia (northwest Spain). In the present study, based on molecular data, a potential P. tapetis specimen was identified from the gill tissues of the mussel Adipicola pacifica associated with whale carcasses (generating chemosynthetic-based ecosystems) collected at shelf depths in the northwest Pacific (southwest Japan). Small subunit ribosomal DNA sequences (1751 sites) of the genotypes of P. tapetis from Spain and Japan were almost identical (only one substitution and one insertion/deletion difference). On the other hand, differences of 10 and 8 substitutions were found in two internal transcribed spacer regions of ribosomal DNA, ITS1 (288 sites) and ITS2 (251 sites) between these two genotypes, respectively, indicating that they are genetically different at the population level. These findings suggest that P. tapetis occurs worldwide and can associate with (and possibly infect) various types of bivalves. Further, a PCR method to specifically detect the P. tapetis cells in the host was also established.     

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.     

Modulatory effects of hard clam (Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

Minchinia nelsoni (Haplosporida) Disease Syndrome in the American Oyster Crassostrea virginica

SYNOPSIS. Minchinia nelsoni disease was studied in 3 populations of oysters from Marumsco Bar, Pocomoke Sound, Maryland. The native population was sampled regularly beginning in 1961 shortly after the disease invaded this area. Two populations of healthy year-old oysters were introduced (one in 1965 and one in 1966) into Pocomoke Sound and studied concurrently with the native population to determine the course of development of the disease and epizootic differences in populations. Gross and microscopic examination of oysters showed progressive stages of infection. Initial infection occurred in epithelia of the filtering organs (gill, palp, water tubes) and spread into connective tissue where hyaline hemocytes infiltrated. Intermediate infection was characterized by local infection and infiltration of connective tissue in and adjacent to epithelia of gill, palp, esophagus, stomach, gut, diverticula, and gonadal alveoli. Advanced infection was recognized by the general invasion and infiltration of connective tissue and the circulatory system by hyaline hemocytes. Terminal infections showed histologically massive pyknosis of nuclei and necrosis of tissues before outward signs of death were apparent. Remission of the disease was recognized by diminution of infection intensity and infiltration; localization of parasites near external epithelia; increased pigment cell formation; and diapedesis and deposition of necrotic parasites and tissue against the shell, followed by external conchiolinous encapsulation. Death from M. nelsoni disease may be attributed in part to seasonal environmental or physiologic stresses, which infected oysters, weakened and in poor condition due to the pathologic manifestations of the disease, are unable to tolerate. The gross pathologic signs of disease were: pale digestive gland, poor condition, mantle recession, weakness, and conchiolinous shell depositions. The microscopic signs were: diapedesis, relative decrease in numbers of phagocytes; relative increase in numbers of hyaline hemocytes; phagocytosis, fibrosis, cellular infiltration, abscess, ulceration, excessive pigment cell formation, mechanical disruption, pyknosis, and necrosis.