Isochrysis

Ripe B. lanceolatum adults were collected by dredging in Argelès-sur-Mer, France, and retrieved from the sand by sieving. Animals were transported, quarantined and maintained in Villefranche-sur-Mer as previously described (Carvalho et al., 2017b (link)). Spawning was induced by a 36-h thermal shock at 23°C (Fuentes et al., 2004 (link)). Sperm and oocytes were collected separately and fertilization was performed in vitro. B. lanceolatum embryos and larvae were raised in the dark at constant temperatures (16°C, 19°C, or 22°C) until the desired developmental stages, and larvae were fed daily with Tisochrysis lutea algae (Carvalho et al., 2017b (link)).
Adult B. floridae were collected in Tampa Bay, FL, United States. Animals were maintained in the laboratory as previously described (Zhang et al., 2007 (link); Yong et al., 2019 (link)). Gametes were obtained either by electric stimulation, heat shock, or spontaneous spawning (Holland and Yu, 2004 (link); Ono et al., 2018 (link)). Embryos and larvae were cultured at constant temperatures (25°C or 30°C) until the desired stages, and larvae were fed daily with Isochrysis sp. algae.
Adult B. belcheri and B. japonicum were collected in Kinmen Island near Xiamen in southeastern China (Zhang et al., 2013 (link)). Animals were maintained as previously described (Zhang et al., 2007 (link); Yong et al., 2019 (link)). Embryos were obtained through spontaneous spawning in the facility (Zhang et al., 2007 (link)). Embryos and larvae were cultured at a constant temperature (24°C for B. belcheri and 25°C for B. japonicum) until the desired stages, and larvae were fed daily with Isochrysis sp. algae.
Asymmetron lucayanum adults were collected in the lagoon between North and South Bimini, Bahamas. Embryos and larvae were obtained and subsequently cultured at a constant temperature (27°C) as previously described (Holland and Holland, 2010 (link)).

Adult M. coruscus mussels were collected at Gouqi Island, (30°72′N; 122°77′E), Zhoushan, Zhejiang Province, China, in August 2016. Upon arrival in the laboratory, the mussels were immediately cleaned and kept in 10-l polycarbonate tanks (∼30 mussels/tank) containing seawater (salinity: 30 ppt) at 27°C, which is the average temperature in the site where the mussels were collected during the summer season. All mussels were fed daily with Platymonas helgolandica var. tsingtaoensis and Isochrysis zhanjiangensis, and the algal density in the tank water was maintained at 2 × 10³ cells⋅ml^-1 and 1 × 10⁴ cells⋅ml^-1, respectively. P. helgolandica and I. zhanjiangensis were chosen due to their tolerance and high growth rate under high temperatures relative to other species (Wang and Wang, 1995 ). The seawater was changed every other day using concentrated seawater from the same location as the mussels (Zhoushan, China) diluted to the appropriate concentration with clean aerated tap water. Mussels were acclimated to the experimental tanks for 1 week before the start of the experiment. The shell length and width of the mussels used for the experiment were 8.8 ± 0.3 cm and 4.3 ± 0.3 cm, respectively.

After transfection, larvae were moved to FSW bubbled with air only for ambient conditions (pH~8.2, pCO₂~400 ppm) or 5% CO₂ and air mixed via multi-gas channel proportioners (Cole Parmer; [76 (link)]) for acidified seawater (pH~7.3, ~3500 ppm) (Table S3). Aquaria were submerged in a water bath held at 25 °C (recommended for oyster larva development; [74 ]). Water changes were performed every other day and larvae were fed ad libitum with cultured Isochrysis sp. and Pavlova lutheri [74 ]. Samples for dissolved inorganic chemistry analysis were collected and read using a VINDTA 3D system, and certified reference material was analyzed during the beginning, middle, and end of runs for quality control (provided by Andrew Dickson, Scripps Institution of Oceanography). DIC, CO₃, pCO₂, alkalinity, and Ω_aragonite, and Ω_calcite were calculated using the seacarb package in R, with known first and second dissociation constants of carbonic acid in seawater [77 (link)].

Seventy-seven ripe adult oysters (4–5 cm long) were collected from floating bags (at East Hampton Shellfish Hatchery, Montauk, NY, USA, on 2 June 2021). Oysters were scrubbed to remove epibionts and sediment and placed in a sea table with raw seawater (17 °C, 31 PSU, pH 7.84) and fed ad libitum with cultured algae (Tetraselmis spp., Isochrysis galbana, Pavlova lutheri, and Chaetoceros muelleri). Seawater was allowed to naturally increase to room temperature (~20 °C, reached within ~1 h). To stimulate spawning, oysters were submitted to a heat shock by adding hot seawater (27 °C) following thermal cycling recommendations of [74 ]. When oysters began spawning, males and females were removed from the sea table, rinsed with seawater and placed in individual aquaria. A subset of oysters were used for strip spawning, which is required for one of the two transfection methods tested (detailed below).

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.