Reproductively sterile fish and shellfish are widely used in aquaculture to reduce ecological concerns associated with aquaculture, improve growth rates and product quality, and allow producers to protect their genetically-selected lines (i.e., intellectual property) from unauthorized propagation. However, current approaches for sterility induction are generally viewed as insufficient. Ploidy manipulation, specifically triploidy, is the most common method employed for sterilization of fish and shellfish. Triploidy induction entails exposing early embryos to a “shock” (e.g., hydrostatic pressure, low/high temperature exposure) to induce retention of a full extra set of chromosomes per cell, changing the ploidy of the individual from diploid (2n) to triploid (3n). Triploid animals generally exhibit reduced survivorship and environmental resilience, which can make them difficult to farm and is of particular concern in light of climate change. Moreover, triploidy methods are underdeveloped for many species and are not always fully effective. Therefore, new approaches for sterility induction are needed.
We are researching an alternative approach for sterility induction that seeks to eliminate the germ cells (i.e., the future gametes) altogether, preventing any possible reproductive development. This novel approach is based on gene silencing technology, which uses “morpholinos” to temporarily silence a gene(s) that is essential to germ cell identity and development. After serving their role, morpholinos degrade and do not introduce any modifications to the DNA (i.e., non-GMO). In order to make this technology scalable for commercial aquaculture, morpholinos were conjugated to a carrier, Vivo, that allows for transport of the morpholinos across the chorion in a bath immersion-style treatment suited for large batches of eggs, circumventing the need to microinject individual eggs/embryos.
Sablefish (Anoplopoma fimbria) and Pacific oyster (Crassostrea gigas ) are being used as model finfish and shellfish aquaculture species, respectively, for this research. In sablefish, proof-of-concept has been achieved. By temporarily silencing a gene known as dead end (dnd ) via immersion of sablefish eggs with a dnd-morpholino-Vivo construct, gonads of some resulting fish were completely devoid of germ cells and did not show any signs of reproductive development. Our future work with sablefish aims to optimize this method to attain higher rates of sterility induction and to evaluate the performance of the sterile sablefish in an aquaculture setting relative to untreated and triploid fish.
Since shellfish do not possess the dnd gene, our research has sought to identify a gene that plays a similar role in germ cell identity/development. Through single-cell RNA sequencing (scRNA-Seq), a cutting-edge approach that uses high-throughput sequencing to identify genes expressed in individual cells, several candidate germ-cell specific genes have been identified in Pacific oysters. These will be the targets for future trials aimed at silencing their expression to potentially induce sterility. Ultimately, we plan to expand this technology to other aquaculture species.