Developing Spermatogonial Germ Cell Transplantation Technology for the Production of Southern Bluefin Tuna (Thunnus maccoyii ) Seed-Producing Surrogates

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doi:10.25907/00447

The Southern bluefin tuna (SBT, Thunnus maccoyii) is one of Australia's most valuable aquaculture species, with total production valued at $153 million in 2012/2013. The aquaculture of SBT is currently based solely on capture of wild fish and as such, it is limited by fishing regulations quotas and seasonal variation in the fish availability. In order to provide sustainable, unlimited supply of SBT, novel and innovative methods are needed to successfully breed it in captivity, despite size and late maturation age limitations. Germ cell transplantation (GCT) is an innovative technology for the production of inter-species surrogates, capable of revolutionizing the aquaculture industry by facilitating easy handling, shorter generation time and a more economical management of large bodied broodstock, such as the SBT. This study aimed at developing the specific methods, molecular tools and knowledge to establish SBT surrogates for the SBT. Yellowtail kingfish (YTK, Seriola lalandi), was assessed as a potential surrogate host for GCT of SBT spermatogonial cells, to produce SBT donor-derived gametes upon sexual maturity. In order to achieve this objective, germ cell populations were described in testis of donor SBT males using basic histology and the molecular markers vasa and dead-end genes. The peripheral area of SBT testis was found to contain the highest proportions of dead-end-expressing transplantable type A spermatogonia (ASG). Percoll-enriched SBT testicular germ cells preparations were then transplanted into the coelomic cavity of 7-9 days post hatch YTK larvae. Fluorescence microscopy and polymerase chain reaction (PCR) analysis have detected presence of SBT cells in the gonads of the transplanted YTK fingerlings at 18, 28, 39 and 75 days post transplantation (dpt), indicating that the transplanted cells migrated to the genital ridge and had colonized the developing gonad. However, further PCR and in situ hybridization analyses of YTK gonads at 165 dpt and PCR analysis of semen collected from sexually mature transplanted YTK males at 1.5 years post transplantation, were not able to detect SBT germ cell-derived DNA or RNA, suggesting that the donor cells were not maintained in the hosts' gonads. This study demonstrates that YTK larvae can support migration and colonization of transplanted SBT spermatogonial cells, despite the phylogenetic distance between the two species. Other species from the Scombridae family (which are phylogenetically closer to SBT) were considered as potential surrogate hosts for GCT of SBT spermatogonial cells, to increase the chances of successful development of the transplanted cells. A method for capture and transport of live pelagic Scombrid fish using "tuna tubes" was developed and optimized. Eight mediumsized mackerel tuna (Euthynnus affinis, also known as eastern little tuna or kawakawa) and leaping bonito (Cybiosarda elegans) were captured and transported alive to the holding tank at the Bribie Island Research Centre (BIRC), 3 of them acclimatized to the tank conditions. Twenty-eight additional small-sized blue mackerels (Scomber australasicus) were captured and transported alive to BIRC, using a 300 L rectangular transport tank, and were treated to induce spawning. None of the captured fish spawned in captivity, but the methods and equipment that were developed and optimized, proved useful for small-scale capture and transport of live Scombrids, as a preliminary step for the establishment of a potential surrogate host broodstock for the SBT. Key genes involved in the reproductive molecular pathway and specifically, germ cell development in the gonads, were described using transcriptome analysis of male and female SBT gonad cells, acquired by next-generation RNA-sequencing. Expression profiles of transcripts from ovary and testis cells were compared, as well as Percoll-enriched cell preparations (as used in GCT) to crude testis cells. ASG-specific genes were identified for the first time in SBT, and were used to develop molecular markers to allow specific identification and localization of different germ cell populations in SBT gonads. These SBT germ cell-specific molecular probes were used to distinguish between transplanted SBT germ cells and the YTK host endogenous cells in a GCT experiment. The methods, experience, tools and knowledge acquired in this study present for the first time the exciting possibility of GCT to produce inter-species surrogates for the SBT. The tools established in this research will enable the Australian aquaculture industry to continue and pursue surrogate technology for the SBT, as well as to other hard-to-breed, large and late-maturing highvalued species.

Developing Spermatogonial Germ Cell Transplantation Technology for the Production of Southern Bluefin Tuna (Thunnus maccoyii ) Seed-Producing Surrogates

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