Combination of indomethacin and temperature produces reliable strobilation in Cassiopea xamachana but batch effects create high variability in ephyra outcomes

Indomethacin and temperature shocks are both widely used to induce ephyra production in Cassiopea polyps, yet the combined effect of these stimuli on strobilation rate, ephyra viability, and polyp survivorship has not been systematically examined. We designed a full factorial experiment crossing five indomethacin concentrations (0, 10, 25, 50, and 75 {micro}M) with four temperature treatments (22, 25, 28, and 31{degrees}C) across three independent experimental trials, exposing a total of 288 individual Cassiopea xamachana polyps for 29 days. Overall, 207 of 288 polyps (71.9%) released at least one ephyra. Temperature was the stronger predictor of strobilation: higher temperatures substantially increased the probability of producing a healthy ephyra (log-odds: +0.327 per {degrees}C; {chi}{superscript 2} p = 6.5 x 10-5), while indomethacin had a smaller but significant positive effect (log-odds: +0.024 per {micro}M; {chi}{superscript 2} p = 0.035). The two stimuli acted independently without significant interaction on healthy ephyra production ({chi}{superscript 2} p = 0.87), although their interaction on total ephyra production (including unhealthy releases) was significant ({chi}{superscript 2} p = 1.0 x 10-9). Notably, low indomethacin concentrations (10 and 25 {micro}M) significantly increased the proportion of unhealthy ephyrae relative to seawater controls (paired t-tests: p = 0.035 and p = 0.006, respectively), while higher concentrations (50-75 {micro}M) did not. Polyp survivorship declined significantly at 28{degrees}C and 31{degrees}C but was unaffected by indomethacin. Trial replicate was the most statistically significant predictor of every outcome measured with the third trial producing near-universal strobilation but zero viable ephyrae, a result of an unidentified perturbation. These results indicate that temperature elevation to 28-31{degrees}C is the most reliable induction strategy for C. xamachana and that low indomethacin concentrations should be avoided when ephyra quality is paramount. We also note that cryptic batch-level variables can overwhelm controlled factors and should be addressed in future experimental designs.