We inoculated aposymbiotic Cassiopea xamachana clones with one of the five genotypes of S. microadriaticum and maintained them at 26°, 30°, and 32°C for 28 days. We also maintained replicates of aposymbiotic polyps at each temperature as a control. For 26 days, we measured the survival of each polyp, as well as asexual reproduction and developmental timing.

After four days of acclimation to temperature (i.e., Day 1), we supplied polyps with access to one of the five genotypes of S. microadriaticum, while also maintaining control polyps with no symbionts. Each well was inoculated with 24,000 cells (2,000 cells/mL) from the appropriate stock algal culture. We fed polyps with Artemia nauplii immediately prior to symbiont inoculation because symbiont uptake occurs more readily when polyps are feeding. We inoculated four six-well plates with each of the five algal genotypes, plus a no algae control, at each temperature, resulting in 24 replicate wells for each genotype by temperature combination (N=432 polyps). We inoculated wells on days 1, 4, 11, 17, 20, and 23 using the same density of cells from the same stock cultures each time. We visually inspected polyps daily under a dissecting microscope to determine survival. When a polyp died, the polyp was removed and the well was emptied. We quantified two strategies of asexual reproduction as the total number of buds and total number of ephyra produced. Buds that were produced during the experiment remained in the well; most buds settled and metamorphosed into polyps, but the experiment was not long enough to allow any newly produced buds to become inoculated and strobilate. We removed all ephyrae that were produced during the experiment the day they detached from the parent polyp. We also measured three developmental timing events: time to inoculation, time to strobilation, and time to ephyra release. Aposymbiotic polyps are white and appear brown once inoculated with algae, so polyps were considered inoculated when a brown tint was observable under the dissecting microscope. Polyps were considered to have begun strobilating when they became disc-shaped rather than cone-shaped. The time to ephyra release was marked as the day that the ephyra detached from the parent bud.