Collection of adults and spawning:  Adults of each species were collected from intertidal or shallow subtidal zones from various sites in Los Angeles County and transported to California State University Long Beach, where they were maintained in recirculating seawater tanks at 16 °C until their use in experiments. Experiments were carried out on one species at a time, depending on reproductive seasonality for that species.

Spawning was induced using standard methods (e.g., M. Strathmann, 1987). The echinoids Dendraster excentricus, Lytechnius pictus, Strongylocentrotus purpuratus and S. fragilis were induced to spawn via injection of 0.2-1.0 mL (depending on adult size) 0.53 M KCL into the perivisceral coelom. The asteroids Patiria miniata and Astropecten armatus were induced to spawn by injection of 1-3 mL 100 µM 1-methyladenine. The holothuroid Apostichopus parvimensis was injected with 3 mL of 200 µM NGLWY-amide (Kato et al., 2009). The ophiuroid Ophiothrix spiculata was exposed to 4 °C water in the dark for 15 minutes, then to room temperature water and sunlight for 15 minutes; this treatment was repeated for up to two hours (Selvakumaraswamy & Byrne, 2000). For all species, adults were each induced to spawn in their own isolated containers, allowing us to control subsequent fertilizations. Spawning continued until a minimum of three parents of each sex were obtained. Sperm from each spawning male was combined with eggs from each spawning female. Once the larvae reached the swimming stage (~24 hours), the offspring of all parents were combined to produce a genetically diverse population.

Culturing: Larvae were distributed into seven replicate 2 l beakers, with a total of 500 larvae per beaker (for S. purpuratus, 14 beakers of 500 larvae each were produced since greater numbers were needed for their protein analysis due to their small size). Larvae were fed 6000 cells ml−1 Rhodomonas lens which were isolated from their growth medium via centrifugation, resuspended in FSW, and counted using a BD Accuri C6 flow cytometer. Cultures were maintained in the 16 °C environmental chamber and continuously stirred by a paddle system (Strathmann, 1987). Daily water changes began on the third day post fertilization (dpf), allowing larvae to develop without disturbance for a day while still in or just completing the pre-feeding period. To change the water, cultures were filtered through a 60 µm sieve to capture larvae. The sieve was submerged in shallow water while filtering so larvae were not exposed to air. Larvae were then gently rinsed from the filter with fresh FSW back into their cleaned beakers and fed.

Sampling: For all species except D. excentricus, sampling for images and protein analysis occurred at 5 (“early-development”) and 10 dpf (“mid-development”). Sampling for D. excentricus occurred at 3 and 5 dpf due to their more rapid development and early formation of the rudiment. While larvae were concentrated during the water change, we collected larvae using a pulled pipette. Depending on larval size and age, three samples of 30-80 larvae each were collected from each beaker and placed in 1.5 ml microcentrifuge tubes on ice. The remaining larvae after the early-development sampling were returned to their beaker and FSW was added to approximate a larval concentration of 0.25 larvae ml−1 based on an estimation of remaining larvae. Both Strongylocentrotus species introduced small variations to these methods, since their small size necessitated experiments with greater numbers. Thus, for S. purpuratus, 14 replicate beakers were made on 1 dpf so three samples of 150 larvae each could be taken on 5 dpf from seven of the beakers (and the remaining larvae in those beakers discarded), and the other seven beakers were used for the mid-development samples of 80 larvae each. S. fragilis was included in this study opportunistically, so we were not able to repeat the experiment with greater numbers and instead had fewer replicates for S. fragilis than for the other species. S. fragilis had a total of 6 replicate cultures instead of 7, and the 10 dpf timepoint consisted of only two protein samples rather than three due to a shortage of larvae.

The tubes containing larvae sampled for protein analysis were centrifuged to pellet the larvae. Water was aspirated gently using a pipette pulled to a tip size of 0.2 mm. Once aspiration was complete, the samples were stored at -80 °C for up to three months.

Protein Quantification: To process the protein samples, we added 100 µl of 4 °C Nanopure water to each sample, and placed them on ice. The larval cells were then lysed using Fisherbrand polypropylene pestles designed to fit 1.5 ml microcentrifuge tubes. A Thermo Scientific Micro BCA Protein Assay Kit was used to determine the protein content of samples against a BSA (Bovine Serum Albumin) standard curve. 376 µl of 4 °C Nanopure water to each to provide adequate volume for the assay. The samples were vortexed for 10-15 seconds. 150 µl from the homogenized sample was then placed into three experimental wells of a 96-well plate to produce three technical replicates per sample. After addition of reagents and incubation according to the manufacturer’s instructions, the plates were read using a Biotek Synergy H1 Hybrid Microplate Reader. To calculate protein content per individual, the number of larvae per technical replicate was calculated as: larvae in technical replicate = larvae in original sample *  (150 µl technical replicate volume)/(476 µl sample volume).