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Alignment of translated open reading frames of a diverse set of insulin sequences identified in the venom glands of various cone snail species. Sequence logo and approximate location of the insulin A and B chain and C-peptide regions are depicted above and below the alignment, respectively (generated in Geneious software, version 8.1.3). Average sequence identities (%) are provided for each region. Cysteines are highlighted in yellow; negatively and positively charged amino acids are shown in red and blue, respectively.

Principal components analyses (PCA) of chemical characteristics of Conus venom and signaling insulins (A), and of venom insulins from cones and signaling insulins from putative prey species (B). Principal components were calculated from the correlation matrices of sequence characteristics using the prcomp package in R (R Core Team 2013). Ninety-five percent confidence ellipses for bivariate plots of the first two principal components were calculated using the data ellipse function in R. Black arrows point to snail-like insulin sequences from C. geographus. These two sequences and one sequence obtained from C. varius (gray arrow) were excluded from ellipse calculations. FH, fish-hunters (yellow); SH, snail-hunters (light blue); WH, worm-hunters (magenta); FP, fish prey (red); SP, snail prey (green); WP, worm prey (blue).

The venom insulins of worm-hunters do not group with worm signaling insulins, but instead with molluscan insulins (both venom and signaling). One potential explanation is that the venom insulins of worm-hunters are used mainly in defense against other mollusks rather than in predation on worms. Interestingly, worm signaling insulins appear to be more diverse than those of fish and snails (fig. 6B). Thus, the insulins of worms actually preyed on by Conus species could differ considerably from those used in this analysis, which were collected opportunistically from the public databases. Future functional studies of venom insulins isolated from worm- and snail-hunting cone snails should reveal their real targets.

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