World Aquaculture Magazine - June 2020

56 JUNE 2020 • WORLD AQUACULTURE • WWW.WA S.ORG In an effort to discover novel means of disrupting PGC development, a small molecule screen was conducted with developing zebrafish embryos expressing green fluorescent protein (GFP) in their PGCs (Fig. 9). Of 7,000 compounds screened, a novel compound called primordazine was identified that specifically blocks the maintenance of PGCs (Jin 2018). In primordazine-treated zebrafish embryos, PGCs are initially specified but rapidly disappear, while other cell lineages appear unaffected. Treated embryos grow to adulthood but are heavily skewed toward males (Jin 2018). The mechanism of action of primordazine is mediated through a short, 40-nucleotide sequence in the 3’ untranslated region (UTR) of the nanos3 gene, a sequence called the primordazine response element (PRE). Through the PRE, primordazine alters the localization and translation of nanos3 RNA in germ cells and, because nanos3 is essential for germ cell maintenance, primordazine ablates PGCs. Interestingly, dissection of primordazine’s mechanism of action revealed that nanos3 , dnd and other early germ cell genes are translated through a non-canonical, polyA-independent form of translation, which is disrupted by primordazine treatment (Jin 2018). The discovery of a non- canonical form of translation that functions during early germ cell development is intriguing because it suggests the existence of novel targets for disrupting germ cell development and sterilizing fish. The studies conducted with zebrafish to date suggest that primordazine-induced ablation of germ cells is not always complete. Often, one or more PGCs survive primordazine treatment and go on to populate the adult gonad. Further studies are necessary to determine whether primordazine treatment alone produces useful sterilization or if primordazine needs to be further optimized chemically or combined with other sterilization techniques. The efficacy of primordazine in ablating PGCs in other fish species also requires further investigation. Investigation of Approaches for Reproductive Sterilization of Sablefish Adam Luckenbach, Edward Hayman, William Fairgrieve, Ten-Tsao Wong, Yonathan Zohar and Thao B. Huynh Escapement is an ecological concern for marine finfishes reared in net-pen systems. Non-native, selectively bred, or genetically modified fish may interbreed with wild stocks, leading to reduced fitness and possible extinction of native populations. The use of reproductively sterile fish is an effective strategy to avoid these effects. In addition, sterile fish may grow faster by minimizing energy allocation to gonadal development and may provide a means for producers to safeguard against unauthorized propagation of proprietary strains. Toward this end, we have initiated research to test three non-GMO approaches for reproductive sterilization: high temperature exposure during early development, triploidy induction and gene silencing technology. Sablefish Anoplopoma fimbria is being used as a marine model species for this line of research because it is an emerging species for net-pen aquaculture in the USA and Canada (Fig. 10) and its sex determination system (XX/XY system; Rondeau et al. 2013, Luckenbach et al. 2017) and early reproductive development (Smith et al. 2013; Luckenbach and Fairgrieve 2016) have been characterized. Temperature is an environmental factor that can influence, redirect or block early reproductive development in fishes. To determine if high temperature exposure during early development could induce sterility, monosex female (XX genotype) sablefish larvae (30 mm) were exposed to elevated water temperatures (20-22 C) for 19 weeks and then transferred to ambient (11.2 C) seawater for one year to determine whether temperature effects were maintained after treatment (Huynh et al. 2019, Fig. 11). In 94 percent of the fish assessed at the end of treatment, ovarian development was markedly inhibited and mRNA levels for germ cell ( vasa , zpc ) and cell death-associated genes ( p53 , casp8 ) generally indicated gonadal degeneration. Gonadal germ cell loss was incomplete however and, after one year at ambient temperatures, ovaries of treated fish exhibited compensatory recovery and were indistinguishable from those of untreated controls (Huynh et al. 2019). Immature testes were observed in 6 percent of the fish. Although elevated temperatures may inhibit ovarian development or trigger sex reversal, this method as administered was ineffective for sterilization of sablefish. In farmed finfish and shellfish species, triploids are often used to avoid problems associated with sexual maturation, including reduced growth, flesh quality and resistance to infectious diseases, as well as to avoid the genetic impact of escaped fish on wild populations. Methods for generating effectively sterile triploids have been developed and proven LEFT: FIGURE 10. Cultured juvenile sablefish from a sterilization trial conducted at NOAA’s Northwest Fisheries Science Center, Manchester Research Station (Port Orchard, Washington, USA). Photo: Edward Hayman. RIGHT: FIGURE 11. Master’s student Thao Huynh records data for juvenile sablefish sampled from a high-temperature exposure study conducted at the Northwest Fisheries Science Center, Manchester Research Station. Photo: Edward Hayman.