Sea Anemone Collection and Culture

Adult Nematostella vectensis sea anemones were collected from the JC NERR near Brigantine, New Jersey, USA in 2020. Spawning was induced approximately twice per month using a standard method for Nematostella vectensis (Hand and Uhlinger 1992; Stefanik et al. 2013). Larval anemones were retained in culture dishes and advanced to the adult stage over 2 years, then spawned in individual cups to identify female and male animals for use in the experiment.

After seven days of acclimation, adult anemones were distributed across 6 tubs (N = 3 tubs per DO treatment: normoxia/control and hypoxia) with 15 females and 14 males in each tub (N = 87 animals per treatment). Next, spawning was induced on day 7 of acclimation to clear anemones of developing gametes (Glass, Schmitt, et al. 2023; Rivera et al. 2021). Total water changes were then performed for each tub, and animals were placed back in ambient culture (i.e., normoxia) for the start of the experimental period (i.e., experimental day 0).

Hypoxia Treatments

Over nights 5, 6, and 7 of the experimental period, adult anemones were exposed to either normoxia (i.e., controls; DO = 7–10 mg L−1) or hypoxia (DO = 0.5–1.5 mg L−1) for ~12 h night−1. Nitrogen gas was used to deoxygenate the water in a glass jar filled nearly to the brim (500 mL; Ball Corporation, Westminster, CO, USA) to a value of ~1.5 mg L−1 (salinity corrected) as determined by a handheld DO probe (YSI ProSolo 626650; accuracy = ±0.02 and precision = 0.01 mg L−1; YSI, Yellow Springs, OH, USA). The anemones from 1 culture tub (N = 29 animals) were transferred with minimal culture water (containing no Artemia) into the jar, which was then topped off with anoxic 12 ppt ASW, capped, and covered with plastic wrap secured by a rubber band. This process was repeated for the 5 other jars, skipping the seawater deoxygenation step for the 3 normoxia jars, and all 6 jars were placed in a dark incubator at 18°C. While anemones were in the treatment jars, the (normoxic) seawater from the culture tubs was pooled and filtered (mesh size 100 μm) before being redistributed across the tubs to minimise block effects by tub and maintain water cleanliness by removing particulate waste, though this may have introduced a confounding factor and should be considered when interpreting the data.

After ~12 h, the jars were opened and a DO reading was quickly recorded. Anemones were then transferred back into their respective tubs with minimal water carried over from the treatment jars. Both animals exposed to hypoxia and controls were handled in the same manner.

Measurements

Detailed methods can be found in the associated publication (Glass et al. 2025).

Dry weight of adult sea anemones was determined using a drying oven and lab balance.
Respiration rates of adult and larval sea anemones were determined via respirometry using a PreSens sensor dish reader system.
Female fecundity was determined by counting eggs imaged via brightifled microscopy.
Male fecundity was determined by counting sperm via flow cytometry.
Egg size was determined by measuring eggs in images collected via brightfield microscopy.
Sperm mitochondrial membrane potential was determined using the fluorescent dye JC-1 analyzed via flow cytometry.
Larval developmental success and settlement rates were determined via visual inspection under brightfield microscopy.