Molecular Characterisation of Immune Genes in Yellowtail Kingfish (Seriola lalandi)
Broomfield, G. R. (2015). Molecular Characterisation of Immune 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/10289/10901
Permanent Research Commons link: http://hdl.handle.net/10289/10901
The effective and efficient introduction of a new candidate species for aquaculture depends greatly on the understanding of the physiology of the organism and the ability to monitor a candidates’ response to changes in their environment. For any farmed fish in an aquaculture setting, there will be a number of bottlenecks that restrict optimal growth and production. Being able to monitor and maintain health is one of these, with stress and pathogenic invasion from microbial organisms leading to a reduction in farming efficiency. Due to the very nature of aquaculture, stress and transmission of pathogens are very common, as fish are contained in close quarters. Thus, characterising and understanding the immune system of these fish will allow for better monitoring of fish health. Yellowtail Kingfish (Seriola lalandi) is a strong candidate for introduction into the New Zealand aquaculture industry. The selection of this fish as a candidate for aquaculture is based on its economically beneficial traits that have potential in both domestic and export markets, particularly in the sashimi market in Japan. Because of this, it is important to begin investigations into the immune response of this species, so aspects of its health can be monitored during farming. The focus of this investigation was to identify immune genes in this species and to begin profiling their expression during the development of the fish. Using bioinformatics approaches, the available gene databases and the implementation of a transcriptomic library prepared from the spleen tissue of S. lalandi, a number of immune genes were identified, which included, Immunoglobulin D (IgD), Recombination Activation Gene 1 (RAG1) and Interleukin-1β (IL-1b). Primers were designed for each gene, to enable confirmation of the gene sequences, by PCR and RACE-PCR. Using this approach, no confirmed sequence was obtained for IL-1b, however sequence was obtained for part of the RAG1 gene and a large part of the IgD gene, including the 3’end. The availability of these immune gene sequences, allowed methods to be developed for looking at gene expression. Primers were designed using the confirmed sequences for RAG1 and IgD, as well as the unconfirmed sequence for IL-1b. These were used in qPCR to examine the expression of each gene during development, within larvae at hatch, 3 dph, 12 dph and 18 dph. Expression of each gene was found to increase by 12 dph, but then decrease at 18dph, with IL-1b showing the highest relative expression. However, no significant difference in expression was seen statistically between any of the time points. Lastly, a protocol was developed for the histological sectioning of larval fish at different stages of development. 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 haematoxylin and eosin (H&E). Developing structures within the larvae, including immune tissues, were identified. Future work will use prepared sections to stain for the immune genes, to gain a better understanding of their role. Discovery of immune genes from kingfish, such as the ones characterised in this investigation, will allow the development of invaluable tools. These tools can be used to monitor the health of fish during both the developmental and adult stages. This paves the way for informative studies that will benefit the future aquaculture of this species.
University of Waikato
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