title: Investigating venom synthesis: exploring the composition, variation and gene expression dynamics of Bitis arietans venom
Creators: name: Currier, Rachel
Publisher: name: University of Liverpool Repository
Year: 2013

description: Snake venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that snakes possess a highly efficient venom production system to maintain their predatory arsenal. The dynamics of venom synthesis and the regulatory mechanisms by which the expression of venom protein-encoding genes is controlled are little understood. The overarching aim of the work described in this thesis was to investigate the dynamics of venom synthesis in terms of the production of venom in juvenile snakes from birth and in the immediate replenishment of depleted venom stores using the African Puff Adder (Bitis arietans) as a model viperid species. We also aimed to investigate the underlying control mechanisms which regulate venom production Initial studies revealed a remarkable degree of intra-species variation in the protein profile, immunoreactivity and enzyme activity of venom between B. arietans specimens originating from different geographical origins across sub-Saharan Africa and Arabia, and within the same geographical origin. Variation was most evident in the snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Our findings are of therapeutic importance as observations could translate into variations in the clinical manifestation of B. arietans envenoming and affect the patient response to antivenom treatment. To monitor the synthesis of venom proteins, we exploited the unusual stability of messenger RNA in lyophilised snake venoms as an alternative source of transcriptionally active mRNA to venom gland tissues, thus avoiding the requirement to sacrifice specimens for transcriptome analysis. Our optimised approach was used to quantitatively track changes in expression of venom protein-encoding genes. Our results showed that the gene expression, protein composition and functional activity of juvenile B. arietans venom did not appear to significantly change over time from birth to four years indicating that some aspects of venom are genetically hard-coded. We also showed that venom resynthesis triggered by venom expulsion peaked between days 3-7 following depletion of venom, with different protein families expressed in parallel. It appeared that venom production in both adult and juvenile specimens occurs very rapidly, presumably to ensure that venomous snakes retain their ability to efficiently predate and remain defended from predators. Our findings suggest that highly regulated mechanisms may be in place to ensure the rapid synthesis of venom. As it appeared that different venom protein families shared similar expression levels during venom replenishment, we investigated whether venom genes also showed similarities in their genomic location, organisation and structure, and regulatory elements responsible for controlling expression levels. We have taken the first steps to begin to investigate the genomic structure and organisation of genes encoding venom protein families expressed in B. arietans venom.