Venomics Reveals a Non-Compartmentalised Venom Gland in the Early Diverged Vermivorous Conus distans

Abstract

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages.

Keywords: conotoxins; defensive venom; evolution; proteomics; transcriptomics.

Figure 1

Proteomics of venom gland sections of C. distans. (A(i)–D(i)) show LC-ESI-MS profiles of the four venom duct sections, with major masses labelled. (A(ii)–D(ii)) show mass distributions for the corresponding sections.

Figure 2

Relative expression levels of dominant masses in C. distans venom gland sections. Comparison of relative expression levels (% total area under the curve) of masses across the four sections of the venom gland. (A) LC-ESI-MS chromatograms of (A) Specimen 1 and (B) Specimen 2 were run for 65 min and 100 min, respectively.

Figure 3

Venom gland transcriptome of C. distans. (A) Expression levels of various superfamilies in the terms of number of transcripts and sequence reads. (B) Highly expressed transcripts (expression level >1% of total reads) in C. distans. The superfamily and predicted cysteine framework are indicated. (C) The distribution of cysteine frameworks in the transcriptome dataset.

Figure 4

Principal Component Analysis (PCA) analysis of Conus planorbis. (A(i)–C(ii)) scores plots indicate the diversity of venom gland segments within the C. planorbis, and (A(ii)–C(ii)) loading plots show families of correlated variables. Specimen 1 includes the highest number of mass peaks (430) among three specimens in PCA. P: Proximal, PC: Proximal Central, PMC: Proximal Middle Central, DMC: Distal Middle Central, DC: Distal Central, and D: Distal.

Figure 5

PCA analysis of venom gland segments of C. distans, C. planorbis and C. geographus. (A(i)–C(i)) PC1 and PC2 scores plots show the separation of venom gland segments in indicated species and (A(ii)–C(ii)) PC1 and PC2 loadings plots show the families of correlated variables. Percentages of variance used in PCA of each species are indicated in the scores plot. P: proximal, PC: proximal central, PMC: proximal middle central, DMC: distal middle central, DC: distal central, and D: distal.

Figure 6

Overview of the major venom components of Conidae. The phylogenetic reconstruction was adapted from Puillandre et al. [14]. Lineages in green indicate mollusc-hunting species and lineages in red indicate fish-hunting species. All other lineages are predominantly comprised of worm-hunters except for divergent species such as Conus californicus, which can prey on fish, worms and molluscs.