World Aquaculture Magazine - June 2020

WWW.WA S.ORG • WORLD AQUACULTURE • JUNE 2020 59 A B C D A B C D A B C D The first strategy disrupts maternally produced RNA that localizes to the germ plasm, a cytoplasmic condensate that instructs primordial germ cell (PGC) fate (Whittle and Extavour 2017). We speculated that loss of single or multiple component(s) of the germ plasm will negate the formation of the PGC, causing embryos to grow into sterile fish. We first created germline mutations in 20 germ plasm genes independently. We than interrogated the effect of losing one functional copy of each of these genes. Decreasing the maternal dose of functional mRNA/ protein decreased the number of PGCs in embryo progeny for all but two hemizygous mutant female lines. Homozygous mutants for nine of the genes targeted were either not viable or sterile, indicating disruption of distinct biological processes (e.g., embryo patterning or in the maintenance of the germ line in adult). These mutant lines could not be used in our approach. On the other hand, homozygous mutant females from the 11 remaining germ plasm genes showed no apparent defect and produced embryos that averaged between 65 and 95 percent PGC reduction. We raised these embryos to adulthood and found a positive correlation between the level of PGCs ablation and the severity of the sterility phenotype. Embryos with low PGC counts developed into sexually delayed male and female with small and immature testis and ovaries respectively, while embryos with no visible PGCs developed into sterile adults with either translucid tube-like testes or string-like ovaries respectively (Fig. 15). We are currently studying the interaction between individual mutations in double knockout lines to further dissect the genetic architecture of PGC development. The maternal effect sterilization strategy described here does not require a treatment to reverse sterility and comprises the steps of 1) breeding a fertile hemizygous mutant male and female fish, 2) selecting a female progenitor that is homozygous by genotypic selection, and 3) breeding the homozygous female progenitor to produce the commercial population of sterile progeny. Our second strategy is designed to produce monosex sterile populations to stack the benefit of sterility with sexually dimorphic performance traits (e.g., body size, color, trophic morphology, feeding kinematics). We first investigated gene mutations in two evolutionary conserved pathways, one governing sex differentiation and the other sex competency. We identified five genes necessary for spermiogenesis and two essential for steroid hormone synthesis causing male sterility and masculinization when disrupted. Double mutant combinations for these genes produce an all-male sterile population. Likewise, we identified three genes whose inactivation causes females to develop atrophic ovaries arrested at a previtellogenic stage or string-like ovaries lacking oocytes. We further identified two genes causing genetic males to sex reverse into females. Double mutant combinations for these gene produce all-female sterile populations. Propagation of the double knockout broodstock lines was achieved via germ cell transplantation from a juvenile mutant donor into a germ cell free wild-type recipient embryo. In the resulting recipient chimera, the mutations are silent as the genes targeted are not expressed in germ cells. With this approach, we generated fertile tilapia broodstock that successfully mass produced a sterile monosex population. Monosex sterility may be best suited for species where only a limited number of broodstock are needed for commercial production of seedstock. We are now implementing the transfer of this strategy to salmonids, where the benefit of monosex female populations is already well established. Notes Tillmann Benfey, Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada. Email: benfey@unb.ca Konrad Dabrowski, School of Environment and Natural Resources, The Ohio State University, Columbus, OH 43210, USA. Email: dabrowski.1@osu.edu Rex A. Dunham, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA. Email: dunhara@auburn.edu Ximing Guo, Haskin Shellfish Research Laboratory, Department of Marine and Coastal Sciences, Rutgers University, Port Norris, NJ 08349, USA. Email: xguo@hsrl.rutgers.edu Anita M. Kelly, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Alabama Fish Farming Center, Greensboro, AL 36744, USA. Email: amk0105@auburn.edu Xavier C. Lauth, Center for Aquaculture Technologies, San Diego, CA 92121, USA. Email: xlauth@aquatechcenter.com . The work described here was supported by the Agriculture and Food Research Initiative Competitive Special Grants Program no.2019-67030-29002 and by the Biotechnology Risk Assessment FIGURE 15. Dissected gonads of fertile and sterile Nile tilapia. Female (B) and male (D) with genome edited changes show string-like ovaries and translucid testes devoid of oocytes and spermatozoa, respectively. Age matched control female (A) and male (C) with mature gonads. Gray arrow heads point to gonads from fertile fish and white arrow heads point to the gonad from sterile fish. ( C O N T I N U E D O N P A G E 6 0 ) We found a positive correlation between the level of PGCs ablation and the severity of the sterility phenotype. Embryos with no visible PGCs developed into sterile adults with either translucid tube-like testes or string-like ovaries.