Corals were collected from Narragansett Bay, Rhode Island, USA. Experiments were performed at the University of Rhode Island, and samples were processed and data collected at the University of Pennsylvania.

Adult Astrangia poculata (Ellis & Solander, 1786) colonies (figure 1a of Glass et al., 2023) were collected from Narragansett Bay, Rhode Island, USA (41.49231, -71.41883) in early August 2021 and 2022 and transported in seawater to the University of Rhode Island (URI). Corals were induced to spawn by increasing the temperature from approximately 22 degrees Celsius (°C) to 31°C over 30 minutes and by physical touch. Once spawning began (figure 1b) of Glass et al., 2023), males were moved to individual glass bowls containing 50 milliliters (mL) seawater and left unperturbed until spawning activity ceased (figure 1c of Glass et al., 2023). The seawater containing sperm (i.e. 'sperm water') was then passed through a 100 micrometer (μm) cell strainer. For in vivo assays, sperm from each male were concentrated separately by centrifugation at 1500 × g for 5 minutes. The supernatant was removed, additional sperm water from the same male was added atop the sperm pellet, and centrifugation was repeated to form a large sperm pellet. Following this process, the supernatant was removed and sperm were resuspended in approximately 15 mL sodium-free seawater (NaFSW), a seawater substitute with the same salinity as seawater but lacking sodium ions (Na+). The absence of Na+ in NaFSW prevents the outward flux of H⁺ through an Na+/H+ exchanger and thus facilitates cytosolic alkalinization following the addition of NH₄Cl. Sperm in NaFSW were used for all in vivo assays within 2.5 hours after spawning. Since coral sperm capacity for motility can decline over time following spawning, each pool of sperm in NaFSW was tested for motility capacity immediately prior to use in each in vivo assay by stimulating a small aliquot of the pool with 20 millimolar (mM) NH₄Cl; all sperm pools had at least 50% motility as assessed via phase contrast microscopy (Nikon Eclipse Ni-U microscope and digital camera system DS-Ri1).

Assessment of in vivo sAC activity:
Within 0.5 to 1.5 hours post-spawning, pools of sperm in sodium-free seawater (NaFSW) (N = 3 pools; 1 pool per male) were divided in half and incubated with either dimethyl sulfoxide (DMSO; 0.5% v/v) as a carrier control or the sAC inhibitor KH7 (50 micromolar (µM) in DMSO; Tocris Bioscience) for 30 minutes, then aliquoted into a 96-well plate. For each sperm pool, half of the replicate wells of sperm from each treatment (DMSO or KH7) were amended with either 100 mM NH₄Cl to a final concentration of 20 mM NH₄Cl (stimulated) or an equivalent volume of NaFSW (unstimulated). Triplicate wells per treatment and stimulation condition were lysed at time = 0, 0.1, 0.5, 1, 2, and 5 minutes with the addition of HCl (0.167 M final concentration) with 0.01% (v/v) Triton-X. Cyclic adenosine monophosphate (cAMP) was quantified in each sample well using an enzyme-linked immunosorbent assay according to manufacturer's instructions (ArborAssays K019). Data were analyzed using a four-parameter logistic curve of cAMP standards to determine the nanomoles (nmol) of cAMP in each well, which was normalized to nanograms (ng) of protein in the well determined via Bradford assays.

Assessment of in vivo PKA activity:
Protein kinase A (PKA) activity was assessed in vivo to test the hypothesis that PKA is activated downstream of sAC following cytosolic alkalinization in sperm from A. poculata. Within 1.5 to 2 hours post-spawning, pools of sperm in NaFSW (N = 3 males; 1 pool per male) were divided into three aliquots and treated with either DMSO (0.5% v/v), the sAC inhibitor KH7 (50 μM in DMSO), or the PKA inhibitor H-89 (20 µM in DMSO; Cell Signaling Technologies) for 30 minutes. Sperm from each treatment were then aliquoted into each of six 1.5 mL tubes. One tube from each treatment was flash-frozen after the addition of NaFSW as an unstimulated control. Then, each of the remaining tubes was stimulated with the addition of a stock solution of 100 mM NH₄Cl (20 mM NH₄Cl final concentration). One tube per treatment was then flash-frozen in liquid nitrogen at either 0.1, 0.5, 1, 2, or 5 minutes post-stimulation. Later, proteins were extracted from each sample, quantified via Bradford assays, and run in gel electrophoresis. Following transfer, membranes were probed with a commercial primary antibody recognizing phosphorylated substrates of PKA (Abcam). All blots were developed with pico chemiluminescence reagents (Thermo Fisher Scientific) and imaged on an Amersham Imager 600 (General Electric) with automatic exposure. Total band intensity was quantified for each lane and normalized to the time = 0 lane for each blot in ImageJ.

Assessment of sperm motility:
Sperm motility was assessed within 0.5 to 2.5 hours of spawning under various conditions to determine the roles of sAC and PKA in motility. Videos of sperm in seawater or NaFSW were recorded before and immediately after stimulation with 20 mM NH₄Cl. For videos of sperm in seawater, sperm were never transferred to NaFSW and sat in seawater for up to 2.5 hours, so motility results of sperm in seawater should be interpreted with caution as motility may have declined over time. Next, sperm in NaFSW were amended with either DMSO (0.05% v/v), KH7 (50 µM in DMSO), or H-89 (20 µM in DMSO) and incubated for 30 minutes, and then stimulated with 20 mM NH₄Cl and immediately videoed under a phase contrast microscope (Nikon Eclipse Ni-U microscope and digital camera system DS-Ri1). This experiment was repeated using sperm from each of three males. In all movies, most non-motile sperm could be seen twitching, indicating viability. Each movie was renamed with a random string of characters and corresponding treatment information was recorded in a spreadsheet, then movies were scored by an observer blinded to the treatment as 0, 20, 40, 60, 80, or 100% motile according to the percentage of sperm displaying progressive, directional motility. Motility scores for each treatment were averaged for each male (technical replicates) and then across the males (biological replicates).

920623_v1_motility_assays.csv