Adult mussels (M. coruscus) were obtained from Gouqi Island, Zhejiang Province, China, and immediately transported to the laboratory on the same day. They were cleaned and cultured in a 10 L polycarbonate tank containing filtered (pore size: 0.45 μm) seawater (FSW) passed through a 1.2 μm acetate fiber filter, maintained at 21 °C, and fed a mixed diet of Platymonas helgolandica and Isochrysis zhanjiangensis every day.
Spawning was induced in the laboratory according to a previously described method, with slight modifications [49 (link)]. Briefly, mussels were placed in a sealed bag, which was incubated overnight on ice. Subsequently, they were transferred to a 2 L glass beaker containing FSW to release sperm and spawn. Fertilization was performed by gently mixing eggs and sperm suspensions in FSW and incubating for 20 min. Fertilized eggs were kept at 18 °C in FSW after removing excess sperm by washing with FSW on a nylon plankton net (20 µm mesh size). After 2 days, swimming larvae were fed P. helgolandica and I. zhanjiangensis at a density of 5 × 104 cells/mL daily. Pediveliger larvae were collected, frozen in liquid nitrogen, and stored at −80 °C. These larvae were used to induce metamorphosis, as well as for NO content determination, RNAi, and gene quantification experiments. All animal handling procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University.
Spawning was induced in the laboratory according to a previously described method, with slight modifications [49 (link)]. Briefly, mussels were placed in a sealed bag, which was incubated overnight on ice. Subsequently, they were transferred to a 2 L glass beaker containing FSW to release sperm and spawn. Fertilization was performed by gently mixing eggs and sperm suspensions in FSW and incubating for 20 min. Fertilized eggs were kept at 18 °C in FSW after removing excess sperm by washing with FSW on a nylon plankton net (20 µm mesh size). After 2 days, swimming larvae were fed P. helgolandica and I. zhanjiangensis at a density of 5 × 104 cells/mL daily. Pediveliger larvae were collected, frozen in liquid nitrogen, and stored at −80 °C. These larvae were used to induce metamorphosis, as well as for NO content determination, RNAi, and gene quantification experiments. All animal handling procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University.
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