Adult culture and spawning

Adult Nematostella vectensis sea anemones were collected from a salt marsh in Brigantine, New Jersey in the fall of 2020, and spawning was induced. Larvae (n = 1 cohort with mixed parentage) were then cultured to the planula stage (3 d post-fertilization) for experimentation. An aquarium population of adult Galaxea fascicularis colonies (n = 9 females, 10 males) spawned during August 2023 at Carnegie Science (Baltimore, MD, USA), yielding a cohort of planulae (n = 1 cohort with mixed parentage) that were used in experiments within 48 h. Adult Porites astreoides colones (n = 20) were collected in Bermuda (32°22′13″N, 64°44′27″W) during July 2023. Brooded planulae (n = 4 cohorts) were collected and maintained in culture for ~ 48 h.

At the time of use in experimentation, larvae from all 3 species were in the planula stage and would become competent to settle within 72–96 h (Hand and Uhlinger 1992; Goodbody-Gringley et al. 2018; Wei et al. 2023). Artificial seawater was used for culturing and experimentation for N. vectensis and G. fascicularis, while flow-through, natural seawater facilities at the Bermuda Institute for Ocean Science were used for P. astreoides.

Dissolved oxygen treatments and larval sampling

Stage-matched, swimming planulae of Nematostella vectensis, Galaxea fascicularis, and Porites astreoides (N = 1,200–2,400 larvae species^-1) were divided into six replicate groups (three normoxia/control and three hypoxia) and exposed to 6 h of normoxia (dissolved oxygen (DO) = 6.8–8.69 mg L^-1) or severe hypoxia (DO = 1.58–1.8 mg L^-1; seawater deoxygenated using N₂ gas) inside sealed glass jars (500 mL) overnight from 21:00 h to 03:00 h the following day. The jars were placed at the ambient culture temperature for each species (N. vectensis: 18°C; G. fascicularis: 27°C; P. astreoides: 28°C). At the end of the treatment period, the jars were uncapped and groups of 20–30 larvae (N = 60–90 larvae treatment^-1 species^-1) were transferred to 1.5 mL tubes without seawater for storage at -80°C until processing for targeted metabolomics as described below.

Targeted metabolics via liquid chromatography-mass spectrometry

Groups of frozen larvae were thawed on ice and homogenized in 160 µL of 50:50 0.3% formic acid/acetonitrile in tough microorganism tubes (Revvity, Waltham, MA, USA) at 4°C in a Precellys homogenizer (Bertin Technologies, France). Aliquots of homogenates (20 µL) were extracted with organic solvents for individual targeted liquid chromatography-mass spectrometry (LC-MS) metabolomics assays (acylcarnitines, amino acids, organic acids, and nucleotides). A 10 µL aliquot of each homogenate was also used for protein concentration determination (to normalize metabolite concentrations) via the Bradford method using a bovine albumin serum standard curve. Quantitation of metabolites was achieved using multiple reaction monitoring of calibration solutions and study samples on an Agilent 1290 Infinity UHPLC/6495 triple quadrupole mass spectrometer at the University of Pennsylvania Metabolomics Core (RRID:SCR_022381). Raw data were processed using Mass Hunter quantitative analysis software. Calibration curves (R² = 0.99 or greater) were fitted with a linear or a quadratic curve with a 1/X or 1/X² weighting.