Crassostrea gigas exposure to the dinoflagellate Prorocentrum lima: Histological and gene expression effects on the digestive gland.

Bivalve mollusks bioaccumulate toxins via ingestion of toxic dinoflagellates. In this study, Crassostrea gigas was used to investigate the effects related to Prorocentrum lima exposure. Oysters were fed with three diets Isochrysis galbana (2 × 10(6) cell mL(-1)) control treatment; algal mix of I. galbana (2 × 10(6)) and P. lima (3 × 10(3) cell mL(-1)); and P. lima alone (3 × 10(3) cell mL(-1)). Feeding behavior changes, histopathological alterations, and expression patterns changes of genes involved in cell cycle (p21, cafp55, p53), cytoskeleton (tub, act), and inflammatory process (casp1) were evaluated. Results indicated that the presence of diarrheic shellfish poisoning by P. lima cells decreased the clearance rate (p < 0.05), induced structural loss, significantly decreased tubule area of the digestive gland (p < 0.05), and up-regulated in expression all gene (p < 0.05), suggesting that toxic cells might trigger inflammatory tissue process, disturb cell cycle and cytoskeleton representing a risk to oysters integrity.

Effects of in vitro exposure to diarrheic toxin producer Prorocentrum lima on gene expressions related to cell cycle regulation and immune response in Crassostrea gigas.

BACKGROUND: Crassostrea gigas accumulates diarrheic shellfish toxins (DSP) associated to Prorocentrum lima of which Okadaic acid (OA) causes specific inhibitions of serine and threonine phosphatases 1 and 2A. Its toxic effects have been extensively reported in bivalve mollusks at cellular and physiological levels, but genomic approaches have been scarcely studied. METHODOLOGY/PRINCIPAL FINDINGS: Acute and sub-chronic exposure effects of P. lima were investigated on farmed juvenile C. gigas (3-5 mm). The Pacific oysters were fed with three dinoflagellate concentrations: 0.3, 3, and 30 ×10(3) cells mL-1 along with a nontoxic control diet of Isochrysis galbana. The effects of P. lima on C. gigas were followed by analyzing expression levels of a total of four genes, three involved in cell cycle regulation and one in immune response by polymerase chain reaction and real time quantitative PCR, where changes in time and cell concentration were found. The highest expression levels were found in oysters fed 3 × 10(3) cells mL-1 at 168 h for the cycle regulator p21 protein (9 fold), chromatin assembly factor 1 p55 subunit (8 fold), elongation factor 2 (2 fold), and lipopolysaccharide/ß-1, 3 glucan binding protein (13 fold above base line). Additionally, the transcript level of all the genes decreased in oysters fed wich the mixed diet 30 × 10(3) cells mL-1 of dinoflagellate after 72 h and was lowest in the chromatin assembly factor 1 p55 subunit (0.9 fold below baseline). CONCLUSIONS: On C. gigas the whole cell ingestion of P lima caused a clear mRNA modulation expression of the genes involved in cell cycle regulation and immune system. Over-expression could be related to DNA damage, disturbances in cell cycle continuity, probably a genotoxic effect, as well as an activation of its innate immune system as first line of defense.

Genomics study of the exposure effect of Gymnodinium catenatum, a paralyzing toxin producer, on Crassostrea gigas' defense system and detoxification genes.

BACKGROUND: Crassostrea gigas accumulates paralytic shellfish toxins (PST) associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. PRINCIPAL FINDINGS: We found that 24 h feeding on toxic dinoflagellate cells (acute exposure) induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD), cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450), intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP), and stress responses (glutamine synthetase, GS) in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×10³ cells mL⁻¹) showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×10³ cells mL⁻¹ produced a significant increase on the transcription level than in a mixed diet (3×10³ cells mL⁻¹ of G. catenatum+0.75×106 cells mL⁻¹ I. galbana) in all the analyzed genes. CONCLUSIONS: Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to stress generated by toxic dinoflagellate exposure.