Cloning and characterization of neoplasia-related genes in flat oyster Ostrea edulis.

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters Ostrea edulis in Galicia (NW Spain). Previous research using suppresive substraction hybridisation that had been performed addressing the molecular basis of DN as well as the induction and development of the disease in oysters, yielded the whole open reading frame of nine genes: XBP-1, RACK, NDPk, C1qTNF, RPA3, SAP18, p23, ubiquitin and ferritin. These nine genes were characterized in this study. The phylogenetic relationships for each gene were studied using minimum-evolution methods. Quantitative-PCR assays were also developed to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, disseminated neoplasia, both diseases and in non-affected oysters (control). The expression of XBP-1, NDPk, RPA3, SAP18 and ferritin increased in haemolymph cells of oysters with heavy bonamiosis. The expression of C1qTNF; SAP18 and p23 increased in haemolymph cells of oysters with DN. The expression of XBP-1, RACK, NDPk, RPA3 and p23 significantly increased in haemolymph cells of oysters affected by both diseases. There were changes in the expression of a number of genes in different organs depeding on disease stage: RACK expression increased in gills of oysters with bonamiosis, XBP-1 increased in mantle and digestive organs of oysters with light DN and RPA3 expression increased in gonads of oysters with heavy bonamiosis and heavy neoplasia.

Identification of relevant cancer related-genes in the flat oyster Ostrea edulis affected by disseminated neoplasia.

Disseminated neoplasia (DN), an oyster disease resembling leukaemia, has been reported in a number of species of marine bivalve molluscs. The disease is characterised by a proliferation of abnormal circulating cells of unknown origin resulting in the invasion of tissues and organs, frequently with a fatal end of the affected individuals. To obtain a more comprehensive view of bivalve cancer processes, suppressive subtracted hybridisation (SSH) and quantitative RT-PCR (q-PCR) approaches were combined to investigate changes in the transcriptome of Ostrea edulis haemolymph cells associated to DN. Two SSH libraries were constructed and 587 expressed sequence tags (ESTs) were sequenced, obtaining 329 ESTs which showed expression changes in neoplastic process. Transcription expression analyses (q-PCR) were done for a total of 24 genes that could be relevant in neoplastic process, including genes with role in the regulation of cell cycle, apoptosis or chromosomal defects. Most of those genes had not been reported in association with cancer in non-vertebrate organisms. The over-expression and under-expression of some of those genes in DN-affected oysters was in agreement with observations in vertebrate cancer. The results herein reported contribute to cancer understanding in bivalve molluscs.

Disseminated neoplasia in cockles Cerastoderma edule: ultrastructural characterisation and effects on haemolymph cell parameters.

Disseminated neoplasia (DN) has been detected in cockles from various beds in Galicia (NW Spain). A study was performed to characterise cockle neoplastic cell ultrastructure and to evaluate the effect of this disease at different severity stages on various haemolymph cell parameters. Examination of cockle neoplastic cells with transmission electron microscopy (TEM) showed round shapes and a lack of pseudopods, a high nucleus:cytoplasm diameter ratio, Golgi complexes, abundant mitochondria, ribosomes, and numerous endoplasmic reticulum tubes and electron-lucent vesicles. Various haemolymph cell parameters (cell mortality, non-specific esterase and lysosome biovolume, reactive oxygen intermediates [ROI] production, phagocytosis ability, intracellular Ca2+ and actin levels) were compared between DN severity categories by flow cytometry; haemocyte mortality, non-specific esterase activities and lysosome biovolume were found to be higher with increasing DN severity. The phagocytic ability of neoplastic cells was sharply reduced with regard to haemocytes. The cytoplasmic-free Ca2+ level was higher and actin content lower in haemolymph cells of diseased cockles compared to unaffected ones. A significant increase in ROI production was detected in later stages of disease progression.

Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule.

Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.