Molecular Characterisation of Sex Differentiation Genes in Yellowtail Kingfish (Seriola lalandi)
Elliott, N. B. (2015). Molecular Characterisation of Sex Differentiation Genes in Yellowtail Kingfish (Seriola lalandi) (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/11474
Permanent Research Commons link: https://hdl.handle.net/10289/11474
Species diversification is crucial for the successful establishment and growth of a finfish aquaculture industry in New Zealand. The yellowtail kingfish Seriola lalandi is a promising candidate species with a fast growth rate, high market value, and is successfully farmed overseas, along with several other Seriola species. To truly domesticate any farmed species, understanding reproduction is vital. Sexual differentiation is a key part of early reproductive development and is characterised by the migration of primordial germ cells through the body to the site of the presumptive gonad, where they proliferate and differentiate into spermatogonia and oogonia. A number of key genes are expressed during sex differentiation, but currently very little molecular research has been conducted on S. lalandi. Using bioinformatics approaches, the available gene databases and the implementation of a transcriptomic library prepared from the gonad, ovary and pituitary tissue of S. lalandi, a number of key sex differentiation genes were identified, which included, Vasa, Amh and Cyp19a1a. Primers were designed to each gene, to enable confirmation of the gene sequences, by PCR and RACE-PCR. Using this approach, no confirmed sequence was obtained for Cyp19a1a, however sequences were obtained for part of the Vasa gene and a large part of the Amh gene, including the 3’end. The availability of these sex differentiation gene sequences allowed expression of Vasa, Amh and Cyp19a1a to be investigated. Primers were designed using the confirmed sequences for Vasa and Amh, as well as the unconfirmed sequence for Cyp19a1a. These were used in real-time PCR to examine the expression of each gene during development, within larvae at hatching, 3 days post hatch (dph), 12 dph and 18 dph. Expression of each gene was found to increase by 12 dph, but then decrease at 18dph, however, no meaningful significant difference in expression was seen statistically between any of the time points. Lastly, a protocol was developed for embedding and sectioning of larvae from a range of different ages. A number of attempts were made to optimise an approach, with fish from 3 dph to 60 dph eventually being paraffin embedded, sectioned and stained with either toluidine blue or hematoxylin and eosin (H&E). A number of regions of interest were described, with one showing good similarity to early gonad formation, detailed in other studies. However, due to technical issues and time constraints, no PGCs or presumptive gonad tissues could be positively identified. Future work will use prepared sections to stain for the sex differentiation genes, to gain a better understanding of their role during development. This study has established a platform for further work on S. lalandi by creating a framework of molecular tools for studying sex differentiation genes and their expression in this species. It has also worked towards refining the histological techniques required to successfully analyse structural development in larvae. This will be valuable for future studies with techniques such as in-situ hybridisation, assisting in the development of tools for the improvement of S. lalandi aquaculture, for applications such as monosex culture and surrogate broodstock technology.
University of Waikato
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