Reproductively sterile shellfish are a market-driven need and ecologically sustainable approach to increasing food production via aquaculture. Current methods for inducing sterility in bivalve shellfish, such as the Pacific oyster Crassostrea gigas, primarily focus on ploidy manipulation; polyploid oysters (reproductively sterile triploids) are commonly used in aquaculture. However, generation of triploids is costly, and polyploidy may have adverse effects on oyster survival in suboptimal growout conditions. A potential alternative approach to the generation of sterile oysters is inactivation of a gene(s) essential for germ cell (future gamete) formation, which has been applied recently in multiple finfish species. Before this approach can be applied in bivalves, several significant challenges must be overcome: 1) characterization of the developmental mechanisms and genes involved in bivalve germ cell fate (‘primordial germ cell (PGC) specification’), and 2) optimization of robust, cost-effective methods to temporarily suppress expression of PGC genes via delivery of gene silencing molecules (e.g., siRNA, morpholinos) to bivalve embryos. The latter is a particularly challenging step for shellfish aquaculture because bivalve embryos are extremely small (<100um), the window for molecule delivery is brief, and the molecule delivery approach must be scalable for use in commercial aquaculture settings.
To address these challenges, our collaborative group has applied single-cell RNA-sequencing to assess PGCs formation during C. gigas embryonic development and identified PGC-specific genes. Our data suggest that PGCs are sequestered during gastrulation and uniquely express a suite of genes that are potential targets for disruption of germ cell formation. Using these gene candidates, we are exploring a variety of gene-silencing methods to assess whether disruption of PGC development can result in adult oysters that do not produce gametes. Additionally, we are optimizing assays for assessing gene expression (e.g. Hybridization Chain Reaction (HCR)) and phenotypic differences between wild-type and “knockdown” oysters. Together, these efforts will generate foundational biological insights and practical biotechnological tools for reproductive control in shellfish aquaculture.