Characterisation of ant venom peptides and proteins

University of Technology Sydney. Faculty of Science.Venom peptides are currently being developed as novel therapeutics and bioinsecticides. Given that ants use their venoms for predation and defence against insects, and other organisms, they are a potential source of these peptides. Although ants represent one of the largest groups of venomous animals, little is known about their venom composition. The present study therefore investigated the peptidome, proteome and transcriptome of a range of poneroid and formicoid ant venoms. Initial experiments sought to confirm the insecticidal and antibacterial activity of whole ant venom using house crickets and minimum inhibitory concentration assays, respectively. Several ant venoms showed significant paralytic and insecticidal activity and others showed antibacterial activity peptides confirming the utility in studying ant venoms. Subsequent experiments investigated the difference in venom composition obtained using differing venom collection methods: manual venom gland dissection or electrical stimulation. The peptide and protein components of the bullet ant (Paraponera clavata) were compared and revealed numerous proteins of which 96 could be assigned a biological function, and 70% of which were common to both collection methods. However, the peptidomic analysis revealed over 300 peptides of which only 30% were common to both collection methods. Therefore, each method reveals a unique set of peptides and proteins. The peptide components of six different ant species were also characterised. The venoms were found to contain between 132–1032 peptides, but the large number of undescribed proteins and peptides highlighted the need for a transcriptomic investigation. Accordingly, an integrated approach using a combination of shotgun proteomics in parallel with Illumina sequencing of the venom gland transcriptome was used to identify toxins in the venom of P. clavata. A BLASTx search of the assembled contigs revealed 354 proteins with homology to existing toxins. Alignments of some of these toxins revealed novel insights into their role in ant venom. A Tox|Note analysis revealed several predicted novel peptide toxins, with some conforming to the conotoxin cysteine frameworks VI/VII and framework I, both of which have yielded therapeutic drug and bioinsecticide leads. The translated transcriptome was then used as a database to query the MS/MS data obtained from the shotgun experiment which identified 44 toxins. Several of these were not identified in the transcriptome BLASTx search, such as δ-paraponeritoxin-Pc1e (formerly poneratoxin). These results reveal the advantages of combining proteomic and transcriptomic methods, and further demonstrates the richness and diversity of ant venoms as potential sources of bioactive compounds