| Meretrix meretrix has been cultured on a large-scale in the southern coastal areas in China. However, serious marine pollution in recent years has resulted in a large amount of losses to the aquaculture industry, especially the safety problems caused by pathogenic bacterium and heavy metals, thus resulting in hindering the sustainable development of shellfish culture. Moreover, due to the lack for information of genomic resources, limited researches about the toxicology mechanism caused by marine pollutions has been carried out. In this work, the expression of ferritin, the lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF), the inhibitor of NF-kB (IkB), metallothionein, and glutathione peroxidase were assessed at transcriptional level in order to investigate the toxicological and immune mechanism of the hard clam Meretrix meretrix following challenge with iron, cadmium and bacterium (Vibrio parahaemolyticus) respectively.Fe supplemented in natural seawater led to the increment of Fe content in both hepatopancreas and gill tissue of M. meretrix between4to15days after4.0mg/L Fe exposure. Similarly, the total Cd content in hepatopanceas, kidney and gill tissue et al. increased corresponding to increased Cd concentration in seawater after7days of exposure. Moreover, the glandular epithelium of the digestive tubes showed a gradual hypertrophy and hyperplasia and disruption of epithelial cell lining after7days exposure in cadmium. These results indicated that both Fe and Cd were able to bioaccumulate in hepatopancreas and gill tissues and cause tissues abnormalities, resulting in disorder of biophysiology processes. Both real-time quantitative PCR and western blotting results indicated that the ferritin expression in hepatopancreas tissue had a positive correlation with the concentration of dissolved iron in seawater. Ferritin expression presented twice upregulation at96h exposure under4mg/L Fe stess, whereas the other proteins mRNA expression in the two tissues had a similar trend of upregulation, downregulation and then gradual recovery to normal levels. These results suggest that upregulation of transcription factors, ferritin, peroxiredoxins were induced to alleviate the toxicity and injuries caused by excess metals. The correlation between ferritin expression and environmental iron concentration suggests a new perspective for ferritin being a new potential biomarker for monitoring the level of environmental metals.Vibrio parahaemolyticus exposure triggered immune responses with similar trends of upregulation, downregulation and then gradual recovery to normal levels in different tissues. The significant expression post-challenge responded in LITAF and IkB at3h, in ferritin at24h, and in metallothionein and glutathione peroxidase at48h. Moreover, a relatively higher increase of these five proteins mRNA was determined in hemocyte compared to other tissues from M. meretrix except for metallothionein in hepatopancreas tissue and LITAF in gill tissue. Similar expression profile of these five proteins mRNA were observed as well as iron exposure. This behavior might be linked to their specific functions in physiological processes. These results suggest that similar signaling pathways are triggered during iron and V. parahaemolyticus challenge respectively. Here, we indicated that the ferritin of Meretrix meretrix (MmFer) was an intermediate member in the pathway of iron homeostasis and innate immune defense mechanism in M. meretrix.Ferritin cDNA was cloned to deduce specific functional sites. The subunit structure and phylogenetic tree of ferritin was also analyzed for confirming the type and function of M. meretrix ferritin. The results indicated that MmFer shared high similarity with H-type ferritins among mollusks. Our work provides evidences for understanding the toxicological mechanism in marine mulluscs defense against metal and bacterial challenge, thus giveing new insight to disease control and rational use of essential elements for the sustainable culture of hard clams. |
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