Triploidization Disrupts Early Reproductive Development of Females and Alters Phenotypic Sex Ratios of Sablefish Anoplopoma fimbria

Escapement is an ecological concern for marine finfishes reared in net-pen systems. To mitigate this risk, we have initiated research to test several non-GMO approaches for reproductive sterilization , including high temperature exposure, triploidy , and gene silencing technology. Sablefish ( or black cod, Anoplopoma fimbria ) has been used as a marine model species for this line of research based on our understanding of its sex determination system (XX/XY) and early reproductive development. High temperature exposure (20-22 ºC ) over 19 weeks of early development inhibited sablefish ovarian development and induced female-to-male sex reversal in some individuals . However, complete germ cell loss was not observed in any individuals and ovarian development appeared to fully recover within one year post-treatment. Therefore, high temperature treatment may be useful for generating neomale (XX male) broodstock but is unlikely to be an effective approach for sterilization of sablefish. Protocols for induction of triploidy using hydrostatic pressure or cold shock were also developed for sablefish, as well as methods for ploidy determination in whole larvae or blood samples. In brief, pressure shock of 7,000-9,000 psi applied 10 min post-fertilization for 5 min, or cold shock at -1.5 ºC for 60-120 min induced triploidy in a high percentage of fish while minimizing adverse effects on survival. Diploid (control) and putative triploid sablefish were reared until reaching a size (~200 mm in length) at which gonadal development could be assessed by histology and ploidy and genotypic sex determined from blood samples. We found that diploid females had ovaries with well-developed primary oocytes, while triploid females had ovaries that exhibited suppressed development, with mostly empty lamellae and reduced numbers of smaller primary oocytes. Diploid and triploid males had testes that appeared similar, composed of type-A spermatogonia. At one-year post weaning, the gonadosomatic index (GSI) of phenotypic females was reduced by ~10 fold in triploids compared to diploids, while there was no GSI difference between triploid and diploid males. These results indicate that triploidy was especially disruptive to female reproductive development and may be a valid approach for sterilization of monosex female lines. An important next step will be to assess the performance of triploid sablefish aquaculture. Finally, a trial was conducted to test the efficacy of a gene silencing (morpholino oligomer, MO) procedure targeting the germ cell gene, dead end (dnd ). Sablefish eggs, either pre- or post- fertilization, were immersed in a solution containing a dnd-MO-Vivo ( at a concentration of 10 µM for 24 to 48 h) and the fish reared until gonadal development could be assessed by histology. From the pre-fertilization group, 10% of the fish had gonads completely devoid of germ cells, 15% had significantly reduced germ cell counts relative to untreated controls, and 75% had what appeared to be normal gonads. This demonstrated that it is possible to produce germ-cell free sablefish using dnd-MO-Vivo bath immersion. Ongoing work is focused on optimizing this method and assessing performance of these fish.