Sea urchins are valuable models for understanding genetic control of early deuterostome development, and they can be useful for understanding the interactions between genotype and phenotype. Elucidating such interactions requires genetic tools such as CRISPR-Cas9 and RNAi applied to the genome. Genetically modified (GM) embryos are typically produced in relatively small numbers, ranging from dozens to a few hundred. Standardized larval culture techniques designed to rear small numbers of embryos through the larval and settlement stages will be key for tracking genes through adulthood and creating GM breeding lines of sea urchins. Whereas, embryos and larvae of cold-water species of sea urchins have been used in past years, a warm-water species may be more amendable to GM work across laboratories. Lytechinus variegatus is a temperate-tropical species of sea urchin that inhabits the nearshore seagrass communities of the Gulf of Mexico and western Atlantic. The embryos and larvae of L. variegatus can be reared at room temperature (approximately 22°C), and larvae begin settlement within three weeks of fertilization, which is much faster than many cold-water species.
Microculture of sea urchins involves growing small numbers of individual larvae in test tubes until the rudiment forms prior to settlement, followed by growing the metamorphosed juveniles in small containers until they can feed on formulated pellets (ca. 3 mm test diameter). Our laboratories are evaluating methods for microculture of L. variegatus larvae and juveniles in fully synthetic seawater (Instant Ocean). We investigated housing and feeding strategies for rearing ten larvae through settlement in 20 mL glass test tubes. Sea urchin larvae (full siblings) were housed in 20 mL test tubes containing 15 mL of synthetic seawater at 34 ppt at 22°C with 12:12 light:dark photoperiod. In Experiment 1, tubes were divided equally between one of two devices to simulate oceanic waves: a tube rotator or an orbital shaker (n=10 tubes per device). Larvae were fed 16,000 cells/larva/day of a 1:1 combination of green and red unicellular algae (Tetraselmis suicica and Rhodomonas salina, respectively). Survival to 8-arm pluteus with rudiment (ca. 16 dpf) was 67.5% for the rotator and 50% for the shaker. In Experiment 2, the tube rotator was used (n=10 tubes, n=10 larvae per tube) and larvae were fed 24,000 cells/larva/day of a 1:1 combination of green and red unicellular algae (Dunaliella tertiolecta and Rhodomonas salina, respectively). Survival to 8-arm pluteus with rudiment (ca. 16 dpf) was 68%. Settlement success at 30 dpf for surviving larvae was 60%.
Future challenges include improvement of survival and growth demographics of larvae, optimization of feeding strategies, optimization of settlement and metamorphosis, and early growth of juveniles. Successful survival and growth of these early life stages will greatly enhance the utility of the model for both coastal and inland labs. Funding for this work was provided by the NSF EDGE IOS-1923445 and NSF EDGE IOS-2319783.