Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.     

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Putative Phage Hyperparasite in the Rickettsial Pathogen of Abalone, “Candidatus Xenohaliotis californiensis”

Studies on the ecology of microbial parasites and their hosts are predicated on understanding the assemblage of and relationship among the species present. Changes in organismal morphology and physiology can have profound effects on host–parasite interactions and associated microbial community structure. The marine rickettsial organism, “Candidatus Xenohaliotis californiensis” (WS-RLO), that causes withering syndrome of abalones has had a consistent morphology based on light and electron microscopy. However, a morphological variant of the WS-RLO has recently been observed infecting red abalone from California. We used light and electron microscopy, in situ hybridization and16S rDNA sequence analysis to compare the WS-RLO and the morphologically distinct RLO variant (RLOv). The WS-RLO forms oblong inclusions within the abalone posterior esophagus (PE) and digestive gland (DG) tissues that contain small rod-shaped bacteria; individual bacteria within the light purple inclusions upon hematoxylin and eosin staining cannot be discerned by light microscopy. Like the WS-RLO, the RLOv forms oblong inclusions in the PE and DG but contain large, pleomorphic bacteria that stain dark navy blue with hematoxylin and eosin. Transmission electron microscopy (TEM) examination revealed that the large pleomorphic bacteria within RLOv inclusions were infected with a spherical to icosahedral-shaped putative phage hyperparasite. TEM also revealed the presence of rod-shaped bacteria along the periphery of the RLOv inclusions that were morphologically indistinguishable from the WS-RLO. Binding of the WS-RLO-specific in situ hybridization probe to the RLOv inclusions demonstrated sequence similarity between these RLOs. In addition, sequence analysis revealed 98.9–99.4 % similarity between 16S rDNA sequences of the WS-RLO and RLOv. Collectively, these data suggest that both of these RLOs infecting California abalone are “Candidatus Xenohaliotis californiensis,” and that the novel variant is infected by a putative phage hyperparasite that induced morphological variation of its RLO host.     

Infectivity of resting spores of Massospora cicadina (Entomophthorales: Entomophthoraceae), an entomopathogenic fungus of periodical cicadas (Magicicada spp.) (Homoptera: Cicadidae)

Progeny of eastern oyster, Crassostrea virginica, introduced into France in 1992, were reared in IFREMER facilities to test their growth performances. During the summer of 1993, sporadic mass mortalities (80-90%) occurred among C. virginica spat reared in the IFREMER laboratories in La Tremblade (Charente Maritime, France) and Bouin (Vendée, France). Affected oysters presented mantle retraction and deposition of an anomalous conchiolin layer on the inner surface of the shell. The incidence of oysters with gross signs exceeded 80%. No obvious pathogen was identified in soft tissues by histology and transmission electron microscopy (TEM). However, histological examination showed the presence of anomalous basophilic round structures, 0.5-1 microm in diameter, in gill and mantle connective tissues. These extracellular Feulgen-negative structures reacted positively with the von Kossa stain. TEM examination on mantle and gill samples in diseased spat showed that the basophilic bodies consisted of concentric deposits of an amorphous substance interpreted as containing calcium. These observations may indicate that the mineralization process in spat shells was disturbed without exact determination of the cause. Based on the similarities of the gross signs to those reported in juvenile eastern oysters in the United States, we believe that the cause of the mortalities observed in France was probably the Juvenile Oyster Disease. Moreover, we report for the first time the detection of anomalous amorphous structures in gill and mantle connective tissues associated with mortalities and deposition of an anomalous conchioloin layer on the inner shell surface in C. virginica spat.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3)

A SMARTer? cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively.     

近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA

Asian oyster Crassostrea ariakensis is being considered for introduction to Atlantic coastal waters of the USA. Successful aquaculture of this species will depend partly on mitigating impacts by Bonamia sp., a parasite that has caused high C. ariakensis mortality south of Virginia. To better understand the biology of this parasite and identify strategies for management, we evaluated its seasonal pattern of infection in C. ariakensis at two North Carolina, USA, locations in 2005. Small (<50 mm) triploid C. ariakensis were deployed to upwellers on Bogue Sound in late spring (May), summer (July), early fall (September), late fall (November), and early winter (December) 2005; and two field sites on Masonboro Sound in September 2005. Oyster growth and mortality were evaluated biweekly at Bogue Sound, and weekly at Masonboro, with Bonamia sp. prevalence evaluated using parasite-specific PCR. We used histology to confirm infections in PCR-positive oysters. Bonamia sp. appeared in the late spring Bogue Sound deployment when temperatures approached 25 degrees C, six weeks post-deployment. Summer- and early fall-deployed oysters displayed Bonamia sp. infections after 3-4 weeks. Bonamia sp. prevalences were 75% in Bogue Sound, and 60% in Masonboro. While oyster mortality reached 100% in late spring and summer deployments, early fall deployments showed reduced (17-82%) mortality. Late fall and early winter deployments, made at temperatures <20 degrees C, developed no Bonamia sp. infections at all. Seasonal Bonamia sp. cycling, therefore, is influenced greatly by temperature. Avoiding peak seasonal Bonamia sp. activity will be essential for culturing C. ariakensis in Bonamia sp.-enzootic waters.     

Sublethal effects of experimental exposure to mercury in European flat oyster Ostrea edulis: Cell alterations and quantitative analysis of metal

Oysters display a diversity of uptake mechanisms for metallic elements and distribution in the target organs, namely gills and the digestive gland. Various tissues of the flat oyster, Ostrea edulis, were studied following experimental exposure to 0.025 m (5 g l) of mercury, for up to 34 days. All animals survived the treatment. Data indicate Hg accumulation in gill tissue with a maximum concentration of 38.76 g g dry weight after 25 days of exposure. Hg levels were lower in remaining tissues, in which the maximum concentration (18.47 mg g-1 dry weight) was reached after 18 days of exposure. After these times, concentration in both tissues decreased. Results show that oysters can accumulate Hg from the environment, without their survival being affected during the experimental period. Structural alteration of epithelial tissues of gill and digestive gland of flat oyster was comparable with effects described for other metallic elements in bivalve molluscs. Interstitial tissue was disorganized in the digestive gland, and ultrastructural changes in intracellular endomembranes were detected in epithelial cells of the digestive gland after 18 days of treatment. After 25 days, absorptive epithelial cells of gills showed highly dilated, swollen microvilli. These intracellular alterations are parameters of the incipient response to the accumulation of mercury.     

A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.     

Discriminatory predation by three invertebrates on eastern oysters (Crassostrea virginica) compared with non-native Suminoe oysters (C. ariakensis)

Abstract. Diminished populations of eastern oysters Crassostrea virginica in Chesapeake Bay have stimulated proposals to introduce Crassostrea ariakensis from Asia to restore oyster stocks. As part of a program evaluating possible ramifications of such an introduction, we studied how invertebrate predators responded to this non-native oyster. We compared predation activity under laboratory conditions by oyster drills ( Urosalpinx cinerea; Eupleura caudata ) that bore through an oyster's shell and by the seastar Asterias forbesi that pulls shell valves apart. These three predators preyed significantly (p<0.05) more on the familiar C. virginica than on the novel C. ariakensis . We previously reported that five crab species preyed significantly more on C. ariakensis than on C. virginica , with predation by polyclad flatworms similar between oyster species. Thus, the drills and the seastar differed from the crabs and the flatworms in their response to novel prey. When Urosalpinx cinerea was placed in a Y-maze after being held for 40 d with oysters of one species or the other, the drills moved toward C. virginica effluent more than toward C. ariakensis effluent. This response did not depend on the species of oyster the drills had been held with, suggesting that the drills were responding to more familiar infochemicals from eastern oysters than from the non-native oysters.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Extraction of DNA,RNA, and glycogen from oysters

The macromolecules DNA, RNA, and glycogen (CHO)_N were extracted with phenol from eggs, sperm, and the combined gill, mantle, and digestive gland tissues of oysters, using methods effective for the HeLa cells. From the eggs, most of the (CHO)_N and RNA coprecipitated in 20% ethanol whereas the DNA precipitated from 50% ethanol solutions. From the combined tissues all three macromolecules precipitated in 20% ethanol whereas from sperm they precipitated from 50% ethanol solutions. The DNA preparation from sperm was viscous and white but contained mostly (CHO)_N and some RNA. However, the DNA-(CHO)_N-RNA preparation was rendered insoluble in saline by CaPO₄ treatment and was not toxic for mammalian kidney cells in tissue culture. Thus, the methods do not yield pure nucleic acids but appear suitable for attempts to extract infectious DNA from oysters.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.