Normal Clams Research Articles

Gut microbiome composition likely affects the growth of razor clam Sinonovacula constricta

Accumulating evidence has revealed that gut microbiota contributes crucial roles in the nutrient absorption, pathogen defense and growth of aquatic animals. The razor clam (Sinonovacula constricta) is one of the most important bivalve products among fishery trading commodities, and its intestine harbors a diverse microbial community, yet its relationship with the clam growth remains unclear. To address this gap, using 16S rRNA gene amplicon sequencing, we compared the gut bacterial community structures among cohabitating retarded, overgrown and normal clams collected from identically managed ponds. A significant difference in gut bacterial communities was observed between retarded and overgrown clams and between retarded and normal ones. Specifically, compared to retarded clams, overgrown clams had significantly higher proportion of Vibrionaceae and Oceanospirillaceae family and Vibrio genus, while normal clams had remarkably higher proportions of Mycoplasmataceae family and Lysobacter and Mycoplasma genera. Further, a structural equation modeling uncovered that variations in the gut bacterial community were positively correlated to digestive enzyme activities, which subsequently influenced clam growth traits (λ = 0.78, P < 0.001). The retarded and overgrown clams exhibited a more cooperative and better connected gut bacterial interspecies interaction than normal clams. Notably, the distribution and shifts of gut keystone species were closely concordant with clam growth traits. Additionally, the relative abundances of gut bacterial-mediated functional pathways involved in amino acid, carbohydrate, energy, glycan biosynthesis, and lipid metabolism decreased dramatically in retarded clams compared to overgrown and normal cohorts. Based on above findings, we speculated that an altered gut microbiota could be a factor in determining the retardation or overgrowth of S. constricta.

2007년 봄 인천만 갯벌에 있어 폭풍으로 인한 갯벌의 침식작용에 의해 대기 중으로 노출된 바지락 (Ruditapes philippinarum)의 조직병리학적 특성에 관한 연구

In Incheon bay, mass mortalities of Manila clam associated with winter storms have been reported. In the present study we have monitored pathologic condition of the clams stranded on the tidal flats by the winter storms occurred in late March to early April in 2007. The field surveyed indicated that mortality of the Manila clam in the study areas ranged 10-15%. Condition index, a ratio of tissue weight to the shell weight, of the stranded clams was significantly lower than the non-stranded normal clams collected from the same locations (p < 0.05), indicating that the stranded clams were comparatively in poor physiological condition. Perkinsus olseni, the protozoan parasite was observed most of clams used in the analysis and the infection prevalence ranged 77-90%. The infection intensity of P. olseni determined using Ray’s fluid thioglycollate medium (RFTM) cultivation and the 2M NaOH digestion assay indicated that the clams collected during late March and early April in 2007 involved 67,182-1,124,727 P. olseni cells/g tissue. The infection intensity of clams from Gung-Pyeung was significantly higher than the intensities observed from Dae-Bu and Young-Heung (p < 0.05). No clear correlation was found between the infection intensities of P. olseni in the non-stranded normal clams and the stranded clams. The stranded Manila clams were also infected with trematode parasite with the prevalence ranged 5 (Young-Heung) to 12.5% (Dae-Bu). The trematode-infected clams exhibited castrated follicles in the gonad, a typical sign of trematode infection. It was believed that mass mortality of Manila clam observed in this study was associated with the poor physiological condition as indicated by CI, although impacts of the parasite infection cannot be ruled out.

Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561bp) consists of a 498bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic 89WCGPC93 catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.

Mass Culture and Characterization of Tumor Cells From a Naturally Occurring Invertebrate Cancer Model: Applications for Human and Animal Disease and Environmental Health

On the northeastern coast of the United States and Canada, Mya arenaria, the soft shell clam, develops a diffuse, hemopoetic tumor (a fatal leukemia-like cancer) resulting from inactivation of p53-like family member proteins.These malignant cells provide a model for an unrelated set of human cancer cells that are also characterized by mortalin-based cytoplasmic sequestration of wild-type p53 tumor suppressor protein (mortalin is the mitochondrial Hsp70 protein). Here we describe methods for mass culture and long-term storage of tumor cells from this cancer. These are the first successful efforts at maintaining malignant cells from any marine invertebrate in vitro. Following passage (subculture), these cultures undergo transition from primary cultures to non-immortalized cell lines that continue to proliferate and do not re-differentiate the normal hemocyte phenotype. We also characterize normal clam hemocytes and the pathology of cancerous clam hemocytes in vitro and in vivo using light and electron microscopy, cyto- and immunocytochemistry, molecular biology, and a phagocytosis assay. Our protocols provide biomedical and environmental researchers with ready access to this naturally occurring cancer model. We discuss the clam cancer model regarding (a) human health and disease; (b) animal health, disease, and aquaculture; (c) environmental health monitoring; and (d) future research directions.

Genotoxic Stress–Induced Expression of p53 and Apoptosis in Leukemic Clam Hemocytes with Cytoplasmically Sequestered p53

In nature, the soft shell clam, Mya arenaria, develops a fatal blood cancer in which a highly conserved homologue for wild-type human p53 protein is rendered nonfunctional by cytoplasmic sequestration. In untreated leukemic clam hemocytes, p53 is complexed throughout the cytoplasm with overexpressed variants for both clam homologues (full-length variant, 1,200-fold and truncated variant, 620-fold above normal clam hemocytes) of human mortalin, an Hsp70 family protein. In vitro treatment with etoposide only and in vivo treatment with either etoposide or mitoxantrone induces DNA damage, elevates expression (600-fold) and promotes nuclear translocation of p53, and results in apoptosis of leukemic clam hemocytes. Pretreatment with wheat germ agglutinin followed by etoposide treatment induces DNA damage and elevates p53 expression (893-fold) but does not overcome cytoplasmic sequestration of p53 or induce apoptosis. We show that leukemic clam hemocytes have an intact p53 pathway, and that maintenance of this tumor phenotype requires nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes. The effects of these topoisomerase II poisons may result as mortalin-based cytoplasmic tethering is overwhelmed by de novo expression of p53 protein after DNA damage induced by genotoxic stress. Soft shell clam leukemia provides excellent in vivo and in vitro models for developing genotoxic and nongenotoxic cancer therapies for reactivating p53 transcription in human and other animal cancers displaying mortalin-based cytoplasmic sequestration of the p53 tumor suppressor, such as colorectal cancers and primary and secondary glioblastomas, though not apparently leukemias or lymphomas.

Kinetic model of cadmium accumulation and elimination and metallothionein response in Ruditapes decussatus

The aim of the present study was to determine the response of metallothionein (MT) during Cd accumulation and elimination in different tissues of the estuarine bivalve Ruditapes decussatus exposed to two nominal Cd concentrations (4 and 40 microg/L) for 40 d, followed by a depuration period of 50 d. Cadmium was accumulated in all tissues of R. decussatus at both exposure concentrations, and the accumulation was tissue dependent. Use of the kinetic model showed that in the gills and remaining tissues, Cd was assimilated faster at the beginning of the exposure and decreased with time, possibly limited by the diffusion rate of this metal within the cell. In the digestive gland, however, the Cd was continuously accumulated. This could reflect that the Cd uptake rate is considerably higher than the loss rate and, therefore, that this tissue has a higher capacity to accumulate Cd compared to the other two tissues. Moreover, the application of this kinetic model in the different subcellular fractions showed that the bioconcentration factor was significantly higher in the low-molecular-weight fraction (where MT is found), suggesting that this fraction binds Cd faster, with a high uptake rate (K(u) = 32/d), and eliminates this metal more slowly (K(1) = 0.005/d). During the depuration phase, MT decreased simultaneously with Cd elimination in all tissues, although with a shorter half-life. In conclusion, the MT response prevented Cd in the tissues of R. decussatus from interfering in the normal clam metabolism; therefore, MT acts as a detoxification mechanism of Cd.

Neoplasia detection in Macoma balthica from the Gulf of Gdansk: comparison of flow cytometry, histology and chromosome analysis

A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B' and C'. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B' and C' and on the ratio between the fluorescence value of peaks B, C, B' and C' in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C'. Stage 1 was characterised by a major population of cells in peak B' and more than 10% of cells in the C' peak. Stage 2 consisted of a lower percentage of cells in peak B' and more than 25% of cells in peak C'. Stage 3 of the neoplasia was characterised by a further reduction in peak B' and more than 40% of cells in peak C'. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.

Flow cytometric and chromosome analysis of softshell clams, Mya arenaria, with disseminated neoplasia

Studies were carried out to determine the DNA content and genomic characteristics of abnormal cells in the hemolymph of the softshell clam, Mya arenaria , affected with disseminated neoplasia (DN). Normal clam cells had a model chromosome number of 34 with a range of 26–39, whereas DN cells had chromosome numbers ranging from 44 to 80 with a mode of 69. Chromosome spreads of hemolymph cells from DN clams contained acrocentric and telocentric chromosomes which were absent from normal cells. By flow cytometric analysis, the quantity of DNA in normal clam hemocytes was 3.2 pg cell −1 based on comparisons to human lymphocyte and trout erythrocyte standards. The circulating cells of all clams with DN intensities greater than 10% examined by flow cytometry showed distinct DNA aneuploidy. Normal cells were assigned a DNA index (DI) of 1.0; the DN cells had a mean DI of 1.76 with a range from 1.25 to 2.05. There was no evidence of a pentaploid population as seen in the mussel, Mytilus edulis , affected with DN. A significantly higher proportion of the DNA aneuploid cells were in the S phase of the cell cycle than normal cells. Aneuploid cells in G O /G 1 were also larger in size than their diploid counterparts. Both the flow cytometry and the chromosome analysis indicate that there is a quantitative difference in the DNA content of DN cells compared to normal cells.

Serum-cell interactions in transmission of sarcoma in the soft shell clam, Mya arenaria L.

1. 1. Serum proteins from sarcornatous soft shell clams, Mya arenaria L., enhanced transmission of sarcoma. 2. 2. Sarcoma cells were isolated and administered to the recipients at the same cell density in different sarcoma-protein-free diluents: seawater, serum from normal clams, heat-treated sarcoma serum or protease-digested sarcoma serum. 3. 3. Transmission in these groups was significantly slower than in the group where cells were administered in intact sarcoma serum, demonstrating that the tumor promoting factors in the serum were heat-sensitive proteins. 4. 4. Normal hemocytes administered in sarcoma serum caused mortality but not sarcoma transmission, suggesting the presence of cytotoxic factors in sarcoma serum.

Serious mortality in populations of giant clams on reefs surrounding Lizard Island, Great Barrier Reef

A survey of giant clams (family Tridacnidae) at Lizard Island fringing reefs on the Great Barrier Reef in July 1985 indicated that over the previous 6 weeks the combined total mortality for Tridacna gigas and T. derasa was 28% at Watson's Bay (WB) and 20% at the channel between Palfrey Island and South Island (P-S). Sporadic mortalities continued at intervals through to December 1985 when combined total mortalities at WB and P-S had reached 38% and 32%, respectively. By January 1987, mortality for these two species was 54% at WB and 51% at P-S. Deaths were not restricted to a particular size class and the average size of clams that died did not change with time during the study. Mortality rates for clams which had been previously biopsied for gonad condition and/or those induced to spawn with serotonin injection were not significantly different from controls. Distribution of dead clams was random to slightly clumped with respect to alive and dead clams within WB, and random within P-S. Results of heavy-metal analyses of tissues from moribund clams were similar to previous results for normal clams. The histopathology of tissues from six of eight moribund clams revealed an unidentified unicellular organism which was not found in Tridacna spp. from previous or subsequent samples from Lizard Island reefs or from other reef areas.

Unique antigens on neoplastic cells of the soft shell clam [formula omitted][formula omitted

Soft shell clams ( Mya arenaria ) are commonly found in coastal waters of both the eastern and western United States. These invertebrates, which have an open circulatory system, may develop neoplasms of the haemolymph which ultimately kill the host. In this study we have 1) recorded the prevalence of hematopoietic neoplasms (HN) in Mya arenaria within a 50 mile radius of Woods Hole, Massachusetts and 2) utilized cells from one HN bearing clam to generate a series of monoclonal antibodies. Our data show that determinants are expressed on HN cells which are not detected on normal clam haemocytes, suggesting separate ontogenetic pathways of cell differentiation.

STUDIES ON THE LIFE-HISTORY OF ALLOCREADIUM ALLONEOTENICUM SP. NOV. (ALLOCREADIIDAETREMTODA)

Allocreadium alloneotenicum sp. nov. is described from the haemocoel of Limnéphilus sp., caddis fly larvae, from Cape Cod, Massachusetts. The life-cycle is demonstrated both in natural and experimental infections. The normal clam host is Pisidium abditum. Miracidia hatch in the debris from dead larvae or after ingestion and passage in the feces of the snail, Aplexa hypnorum. The process of miracidial peneration was observed. Secretions from the apical gland are histolytic in action, facilitating penetration, while the "penetration" glands produce the cuticula of the sporocyst. Sporocysts give rise to one generation of mother rediae which in turn liberate daughter rediae. Daughter rediae give rise to ophthalmoxiphidiocercariae and also produce occasional rediae. The cercariae penetrate caddis fly larvae (as many as 25 of them being found in natural infections). They mature in the haemocoel and a single worm was found to have laid 1200 eggs. Experimental infections of fish with infected caddis fly larvae were negative.