Mussel Spawning and Larval Culture

Spawning and larvae culture techniques of the hard-shelled mussels (M. coruscus) were performed as previously described [4 (link)]. Briefly, adult mussels (~120) were cleaned and kept in 10 L tanks containing aerated seawater (salinity: 30 ppt; pH: 8.1) after arrival in the laboratory. Mussels were induced to spawn by removing them from seawater and exposing them to air overnight at room temperature. Mussels were transferred back to the 10 L tanks containing aerated, filtered seawater (FSW; acetate–fiber filter: 1.2 μm pore size) at 18 °C. When mussels started spawning, they were gently transferred to individual 2 L glass beakers, and eggs and sperm were collected by filtration. Fertilization was performed by gently mixing equal volumes of sperm and eggs in FSW at 18 °C. Excess sperm was removed after egg fertilization by filtering the mixture through a nylon plankton net (mesh size: 20 μm), and the fertilized eggs were collected and kept in a 2 L glass beaker containing FSW for two days at 18 °C. Larvae were maintained at a density of 5 larvae mL⁻¹ FSW at 18 °C by feeding with the microalgae Isochrysis zhanjiangensis (50,000 cells ml⁻¹). The following developmental stages were collected: (1) trochophore larvae (1 day post-fertilization, dpf), (2) D-veliger larvae (4 dpf), (3) umbo larvae (15 dpf), (4) pediveliger larvae (41 dpf), and (5) juvenile (post-metamorphic, 63 dpf). Samples of the developing larvae were collected by filtering the FSW through a nylon plankton net (mesh size: 40 μm) and rinsing with sterile FSW. The small size of the larvae and juveniles meant that samples for analysis consisted of pools of larvae (containing approximately 300–3000 individuals/sample) that were collected into microcentrifuge tubes and snap-frozen immediately by immersion in liquid nitrogen. For each developmental stage, 5 samples were collected for RNA extraction and sequencing.
All procedures for mussel acclimation and experimentation were authorized by the Animal Ethics Committee of Shanghai Ocean University with the registration number SHOU-DW-2018-013.