Transcriptomic Messiness in the Venom Duct of Conus miles Contributes to Conotoxin Diversity

Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly. Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly. Cone snails are predatory marine gastropods that feed on a variety of prey, including fish and invertebrates. With ∼700 cone snail species described, Conus represents the largest genus of all invertebrate marine animals. The current classification and phylogeny of cone snails are still a matter of debate and are being refined using genomic DNA and radula morphology data (1Bouchet P. Kantor Y.I. Sysoev A. Puillandre N. A new operational classification of the conoidea.J. Molluscan Stud. 2011; 77: 273-308Crossref Scopus (130) Google Scholar, 2Biggs J.S. Watkins M. Corneli P.S. Olivera B.M. Defining a clade by morphological, molecular and toxinological criteria: distinctive forms related to Conus praecellens A. Adams, 1854.Nautilus. 2010; 124: 1-19PubMed Google Scholar, 3Duda Jr., T.F. Bolin M.B. Meyer C.P. Kohn A.J. Hidden diversity in a hyperdiverse gastropod genus: discovery of previously unidentified members of a Conus species complex.Mol. Phylogenet. 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