Molecular Cloning And Functional Analysis Of Genes Involved In Larval Development In Clam Meretrix Meretrix

Bivalve culture is one of most widely practiced mariculture.in China. Seed rearing is a key phase in large scale bivalve culture industry. The developmental status of early larvae would greatly affect the metamorphosis rate, the later growth and survival of bivalve. Investigation of molecular mechanism in larval development is thus important for production practice.Clam Meretrix meretrix is distributed along the coastal and estuarine areas in eastern Asia. It is an important commercial clam widely cultured in the coast of China. In this paper, we studied M. meretrix ferritin (MmeFer), cathepsin B (MmeCB) and caspase genes, which are involved in clam larval shell formation, nutrition, metabolism and apoptosis, respectively. We have cloned the three genes, investigated the temporal and spatial expression profile both at gene and protein level in trochophore (L1), D-veliger (L2), pediveliger (L3) and postlarvae (L4). The potential roles of these proteins were analyzed with specific inhibitors during larval development.Firstly, embryos were found developed into trochophore-like larvae with no shell if cultured at gastrula stage in artificial seawater without iron. Shell-like structures were formed only in the presence of iron. The larvae which had been transferred at L1 stage into ASW developed normal shell. This indicated that iron and iron associated protein are important for larval shell formation. The EST sequence which is homologous with ferritin, which is a principal iron metabolic protein, was selected from the M. meretrix cDNA library. The full-length of ferritin subunit cDNA (MmeFer) was cloned by RACE. The results of real-time PCR revealed that the MmeFer mRNA expression changed before and after the larval shell formation. The result of whole mount in situ hybridization showed that MmeFer was located at the position of shell initiation in trochophore stage, indicating MmeFer plays a role in shell initiation in M. meretrix.The full-length of M. meretrix cathepsin B (MmeCB) cDNA was cloned with 3'and 5'RACE. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. From L2 to L4 stages, MmeCB mRNA was detected in the digestive tract, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in L2 indicating that it is associated with another pathway of nutrient metabolism. Cathepsin B specific inhibitor (CA074Me) was applied to study the function of MmeCB in the growth of unfed larvae. Larval growth was found influenced which support that MmeCB may participate in nutrient metabolism in epidermis. MmeCB protein expression was detected by immunocytochemistry with specific antibody during larval development. In L3 larvae, MmeCB expression can be detected not only in the digestive tract and epidermis near the shell, but also at the bottom of velum, showing MmeCB might play a role in velum degradation during larval metamorphosis. The potential role of MmeCB in hatching and metamorphosis was studied with CA074Me. The results showed that inhibiting the activity of MmeCB can affect the embryo development and larval metamorphosis, suggesting the multifunction of MmeCB during M. meretrix larval development.Degenerate primers were designed according to the conserved sequence of caspase. A cDNA segment was isolated from M. meretrix larvae. The results of caspase in situ detection in larval developmental stages revealed that there were activated caspase from L1 to L3, indicating caspase is involved in the whole developmental process. The main apoptosis area was found in velum at L3 before metamorphosis, indicating the degradation of velum might be the process of apoptosis. The larvae before metamorphosis were treated by caspase specific inhibitor to analyze the role of caspase in this process. The results indicated that caspase have function metamorphosis.In conclusion, the three genes (MmeFer, MmeCB and caspase) associated with larval shell formation from L1 to L2 stage, larval nutrient metabolism from L2 to L3, and metamorphosis from L3 to L4, have been characterized and analyzed respectively. The investigation will be helpful for understanding the molecular mechanism in bivalve larval development.