RNA Interference in Achelatan Lobsters - Exploring and Understanding Mechanisms of Gene Silencing to Promote Aquaculture Biotechnology

Thomas Banks

doi:10.25907/00897

Significant innovations in lobster hatchery technology have led to the emergence of the ornate spiny lobster, Panulirus ornatus, and the slipper lobster, Thenus australiensis as candidate aquaculture species. Unprecedented access to these species across their complex life history has prompted research into developing biotechnological tools and applications to support the growing industry. The use of RNA interference (RNAi) as a molecular tool for functional annotation and generation of desirable phenotypes has great potential to enhance the aquaculture of P. ornatus and T. australiensis, however further research is required to establish this technique in these species. In T. australiensis, gene silencing by RNAi is effective and robust, however a greater number of genetic resources are needed to characterise and identify important genes and pathways which can be silencing targets. In P. ornatus conversely, a wealth of genetic resources has been established, but these cannot be harnessed readily as RNAi historically shows limited efficiency in spiny lobsters, which hinders functional gene annotation. With these challenges in mind, the research presented in this thesis aims to investigate the mechanisms of RNAi induced gene silencing in P. ornatus and T. australiensis and develop the capacity for efficient gene silencing and its application in lobster aquaculture. Initially in T. australiensis, the first comprehensive tissue transcriptome for this species was established, derived from reproductively mature individuals to facilitate identification of RNAi targets for sexual manipulation. Using this data, a neuropeptidome was created, which revealed a number of neuropeptides expressed highly in reproductive tissues. Following this, the insulin-like androgenic gland hormone (IAG) was characterised in silico and in vivo, prior to in vivo silencing for functional annotation. Silencing IAG chronically impaired spermatogenesis, which provides a foundation for future research in manipulating reproduction via RNAi to facilitate desirable phenotypes such as all-female mono-sex populations. Further, the RNAi machinery was compared through transcriptomics in T. australiensis and P. ornatus to better elucidate the mechanisms by which silencing is inefficient in P. ornatus. A comprehensive bioinformatic analysis identified the core RNAi machinery and characterised its expression using multiple transcriptomic databases. In both species, all the machinery in the small interfering RNA (siRNA) pathway was present, however the expression of the key siRNA pathway proteins Dicer 2, Argonaute 2, and SID1 was significantly higher in T. australiensis than in P. ornatus across multiple tissues. The reduced expression of the siRNA machinery provided the foundation of a possible limiting mechanism of RNAi in P. ornatus. Finally, a robust investigation of the mechanisms of RNAi was conducted in P. ornatus across its life history, which revealed significant silencing efficiency in juvenile individuals, and inefficient RNAi in the larvae. The siRNA pathway was assessed in larval and juvenile P. ornatus, where the double stranded RNA (dsRNA) uptake channel SID1 showed insufficient expression exclusively in the larvae. The uptake of dsRNA was investigated further with fluorescent microscopy, which revealed that dsRNA is sequestered in the antennal gland rapidly, likely contributing to reduced RNAi efficiency systemically. Meanwhile in P. ornatus juveniles, extremely efficient RNAi was observed across a number of tissues, which immediately enable functional annotation of important genes and subsequent biotechnology application. In summary, our studies have enhanced the capacity and use of RNAi in the aquaculture of two commercially important lobster species by increasing available genetic resources and molecular tools. This foundation supports the development of RNAi based biotechnology in P. ornatus and T. australiensis aquaculture to promote sustainability and productivity. The mechanisms described within these studies furthermore elucidate the RNAi response in decapod crustaceans, which advances the understanding of the complex and evolutionarily conserved RNAi pathway.

RNA Interference in Achelatan Lobsters - Exploring and Understanding Mechanisms of Gene Silencing to Promote Aquaculture Biotechnology

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