Methods for generating triploid green sea urchin embryos: An initial step in producing triploid adults for land-based and near-shore aquaculture

Here we provide methods for generating triploid green sea urchin embryos (Strongylocentrotus droebachiensis). These are the first triploid sea urchins of any species that have been produced at any life stage and represent an initial step in a larger effort to yield triploid adult green sea urchins for large-scale land-based and near-shore commercial aquaculture ventures. Following fertilization, triploid mollusks and fish result by preventing release of the second polar body. Adult triploids in all of these organisms are sterile and may be larger than diploids based on accelerated growth of somatic tissues. Similar methods are impossible for sea urchins since haploid (1n) ova are released from the ovary after completion of both meiotic divisions. We have produced triploid green sea urchin embryos (3n = 63) by fusing two 1n ova (each n = 21) and fertilizing the diploid products of these fusions with highly diluted spermatozoa (n = 21). Mechanical, proteolytic and acidic treatments were employed to remove the jelly coat and vitelline membrane prior to fusion. Results indicate that acidic removal of the extracellular structures, subsequent fusion with poly(Arg) in 1.5 ml microcentrifuge tubes and fertilization with highly diluted sperm yield 60.64 ± 1.39% triploid embryos that reach prism pluteus stage (with spicules). Triploidy was verified at all developmental stages by counting chromosomes. We discuss scaling up our laboratory methods for embryos to the hatchery environment and also issues unique to the reproductive biology of sea urchins that will be vital for commercial aquaculture ventures to consider if triploid adult green sea urchins can ultimately be produced.