World Aquaculture Magazine - June 2020

WWW.WA S.ORG • WORLD AQUACULTURE • JUNE 2020 55 ( C O N T I N U E D O N P A G E 5 6 ) producers to protect their valuable selected strains from unauthorized propagation (Wong and Zohar 2015a, 2018). We have developed a non- transgenic method to produce infertile fish by irreversibly disrupting the normal migration and development patterns of primordial germ cells (PGCs; Wong and Zohar 2015b). In fish, the PGCs are formed and migrate to the future gonads during early embryogenesis. These germ cells transform into spermatogonia or oogonia and the gonad completes development and forms a testis or ovary in the male or female to produce either sperm or eggs, respectively, upon sexual differentiation and maturation. Of particular interest, we discovered that a molecular transporter comprised of a dendrimeric oligoguanidine with a triazine core, also known as Vivo, can effectively carry the gene silencer morpholino oligomer (MO) across the chorion, enter the embryo and reach the target PGCs. In our new approach, we established a practical protocol to administer MOs into fish embryos using a bath-immersion technology with Vivo-conjugated MOs (Fig. 8). Immersion of zebrafish embryos in the Vivo-conjugated MO against zebrafish deadend ( dnd ) effectively disrupted PGC development. Dnd is an evolutionarily conserved RNA- binding protein that is specifically expressed in PGCs and plays an essential role in their development (Weidinger et al. 2003). Blocking dnd protein synthesis with a dnd MO led to the elimination of PGCs and resulted in the production of reproductively sterile fish that possessed minimally developed gonads without any gametes (Wong and Zohar 2015b). At this time, the technology has been successfully applied to zebrafish and achieved 100 percent sterility induction. Our ongoing experiments in rainbow trout, tilapia, Atlantic salmon and sablefish indicate that this technology can also be applied to induce sterility in commercially important aquaculture species. In addition to finfish, we are also extending this bath-immersion technology to produce sterile oysters by disrupting their early PGC development. Different from current triploid oysters, this alternative sterilization technology will preserve the market advantages of growing sterile oysters without altering their chromosome sets. The abnormality of chromosome sets in triploid oysters is considered to be a cause for greater mortality in triploid oysters, particularly in a subtropical region where environmental conditions can change rapidly and dramatically. Currently, we are continuously optimizing this sterilization technology in several aquaculture species, as well as studying its impacts on growth-related traits by comparing growth performance and fillet yield among sterilized diploids, normal fertile diploids and triploids. Even if sterility is not 100 percent achieved in some cases during commercial operation, this non-transgenic approach, unlike transgenic approaches, carries no risk of transferring transgenic traits into wild populations. It will also eliminate the negative perception of genetically modified aquaculture products that may face long-term resistance from consumers even if their production and marketing is approved by government regulators. Primordazine-Induced Ablation of Primordial Germ Cells Randy Peterson The molecular mechanisms that specify germ cells, maintain their fate, and direct their migration towards the developing gonad involve a complex array of protein-protein, protein-RNA and protein-DNA interactions. Disruption of these processes could induce Primordial Germ Cell (PGC) loss, and without PGCs the embryos would be expected to develop as sterile fish. Therefore, small molecule inhibitors that disrupt PGC development could become effective tools for generating sterile fish. FIGURE 8. A flow chart of pre-fertilization and post-fertilization bath immersion methods to produce infertile fish for aquaculture and fertile fish for broodstocks. dnd -MO-Vivo, administered by bath immersion, disrupts primordial germ cell (PGC, red circles) development and migration at very early stage. In dnd -MO-Vivo treated embryos, PGCs are not able to reach the developing gonads (green), which results in production of infertile fish. Broodstocks can be generated by simply skipping the immersion with dnd -MO-Vivo. FIGURE 9. Schematic of screen for PGC-ablating chemical compounds. A transgenic zebrafish line expressing green fluorescent protein in its primordial germ cells was used to generate >104 embryos, which were arrayed 3 embryos/well in 96-well plates. Compounds from a small molecule library were transferred into the wells, and embryos were inspected under a fluorescent microscope. The screen identified primordazine, which ablates PGCs. Wt miR + Control Wt miR + C6 control primordazine 7,000 compounds