Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss

Abstract

Background: Cephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland.

Findings: To reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non-tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source.

Conclusions: We present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

Keywords: cephalopod genome; comparative genomics; gene family expansions; transposable elements; venom evolution.

Figure 1:

Comparisons of molluscan genomes and gene families. (A) Time-calibrated maximum likelihood phylogeny of 7 molluscan genomes (Aplysia californica, Lottia gigantea, Crassostrea gigas, Euprymna scolopes, Octopus bimaculoides, Callistoctopus minor,andHapalochlaena maculosa) and 4 transcriptomes (Octopus kaurna, Octopus vulgaris, Sepia officinalis, and Idiosepius notoides) using 2,108 single-copy orthologous sequence clusters. Node labels show divergence times in millions of years (mya); blue (divergence to octopods) and orange bars (decapods) represent standard error within a 95% confidence interval. Octopodiformes lineages are highlighted in blue and decapod orange. Scale bar represents mya. (B) Expansions of octopod gene families relative to molluscan genomes Aplysia californica (A. cali), Biomphalaria glabrata (B. glab), C. gigas (C. gig), L. gigantea (L. gig), E. scolopes (E. scol), C. minor (C. min), O. bimaculoides (O. bim), and H. maculosa (H. mac). (C) Lineage-specific gene expansions in the octopod genomes C. minor (C. min), O. bimaculoides (O. bim), and H. maculosa (H. mac). CHGN: chondroitin N-acetylgalactosaminyltransferase; C2H2: Cys2-His2; SPRR: small proline-rich proteins.

Figure 2:

Dynamics of gene expression in octopod genomes. Proportion of gene expression across levels of specificity from not specific to octopods or an octopus species (left) to octopod-specific (middle) and lineage-specific (right). Donut plots show gene expression as some expression in any tissue (purple), no expression (blue), or expression that has been lost (dark blue). Loss of expression requires an ortholog of the gene to be expressed in ≥1 species and not expressed in the other species. Heatmaps at each specificity level show average expression of genes within their respective tissues, low expression (cream) to high expression (dark red).

Figure 3:

Dynamics of gene expression in neural and venom-producing tissues of octopods. Tissue-specific expression of genes within the brain (red) and posterior salivary gland (PSG) (blue) of H. maculosa (H.mac), O. bimaculoides (O.bim), and C. minor (C.min). A) Venn diagram shows numbers of shared and exclusive genes between species (left). B) Bar chart of the top 5 Pfams and their contribution to overall expression in the brain (right).

Figure 4:

Examination of posterior salivary gland (PSG) gene expression between 3 octopod genomes. (A) Heat map of genes expressed specifically in the PSG of H. maculosa (τ > 0.8) and their orthologs in O. bimaculoides and C. minor lacking specific expression to the PSG (τ < 0.8). Genes with an ortholog lacking expression are coloured in grey while the absence of an ortholog is white. (B) Heat map of genes expressed specifically in the PSG of both O. bimaculoides and C. minor (τ > 0.8) and their orthologs in H. maculosa lacking specific expression to the PSG.

Figure 5:

Mechanism of tetrodotoxin resistance within the posterior salivary gland of H. maculosa (PSG). (A) Alignment of voltage-gated sodium channel α-subunits (DI, DII, DIII, and DIV) p-loop regions. Mutations conferring resistance are coloured in green (pufferfish), orange (salamander), purple (clam), and blue (octopus). Susceptible mutations at the same site are black and boldface. Sites that may be involved with resistance are in boldface. (B) Schematic of voltage-gated sodium channel (Na_v) α-subunits (DI, DII, DIII, and DIV). Each unit is composed of 6 subunits, 1–4 (blue) and 5–6 (yellow). Alternating extra- and intracellular loops are shown in black with the p-loops between subunits 5 and 6 highlighted in red. Mutations conferring resistance are shown within black circles on p-loops.

Figure 6:

Assessment of bacteria within the posterior salivary gland of H. maculosa (PSG). (A) Bacterial composition at the phylum level of anH. maculosa posterior salivary/venom gland. (B) Composition of the largest phylum, Protobacteria, of an H. maculosa posterior salivary/venom gland.