Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails

Abstract

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.

Figure 1. The predation- and defence-evoked venoms of cone snails.

(a–c) LC-MS profiles of venom samples collected from the deadly piscivorous Conus geographus following alternating predatory (a,c) and defensive (b) stimuli (milkings separated by 1–7 days). The two predation-evoked venom samples (clear venom), although interrupted by a defensive milking (milky venom with granules), were identical in composition and contained mainly the non-paralytic contryphan-G, conopressin-G, conophysin-G and a new conotoxin (G117). In contrast, the defence-evoked venom was more complex and contained paralytic conotoxins, including the presynaptic calcium channel blockers ω-GVIA, ω-GVIB, ω-GVIIA, the postsynaptic muscle nicotinic receptor antagonists α-GI, α-GIA and α-GII, and the sodium channel inhibitor μ-GIIIA, which evidently are used to defend against predators rather than for prey capture, as previously believed. (d) Overall, <50% of the major predatory toxins are also injected in defence, mostly at significantly lower levels (the number of previously characterized toxins compared with the total number of major masses detected are shown in parenthesis). (e–g) Show similar data for the molluscivorous C. marmoreus, which can repeatedly inject venom over much shorter intervals than piscivorous species (minutes versus days). (h) Remarkably, the predation- and defence-evoked venoms of C. marmoreus are even more divergent compared with the venom of C. geographus. Again, the defence-evoked venom contained vertebrate-active neurotoxins, including χ-MrIA, μO-MrVIA and μO-MrVIB. Examining the number of previously characterized toxins revealed a bias towards the discovery of defensive toxins in previous studies (d,h).

Figure 2. Action of predation- and defence-evoked C. geographus venoms on human receptors.

(a–d) Both predation- (blue) and defence-evoked (green) venoms (1 mg each) were separated on RP-HPLC. The resulting 72 1-min fractions (F1-F72) were screened on SH-SY5Y human neuroblastoma cells for activity at α7 (a,b) and α3-containing nicotinic receptors (c,d), Na_v1.2 and Na_v1.7 voltage-gated sodium channel (e,f) and Ca_v2.2 voltage-gated calcium channel (g,h). Active fractions are highlighted in red on the left panels, with a response ratio >1 indicating greater activity in the defence than the predation-evoked venom, and vice versa (except F32 and F33, which show minor slowing of the response for Nav1.2/7 in both predation- and defence-evoked venoms). Specific responses for active fractions are shown on the right panels (b,d,f and h), and known toxins detected in these fractions are indicated. Whereas the predation-evoked venom only shows full inhibition of Ca_v2.2 response due to trace amount of ω-GVIIA and ω-GVIA, the defence-evoked venom shows potent inhibition of all molecular targets, with several well-characterized toxins identified in the active fractions. The potent block of these key physiological ion channels explains the lethal effect of C. geographus defensive envenomation on humans.

Figure 3. Distribution of toxins in C. geographus venom duct and proposed mechanism for venom release.

(a) Twelve venom gland sections were spotted on a MALDI plate together with predation- and defence-evoked venom (O, oesophagus; P, proboscis; RS, radular sac; SG, salivary gland). (b) The resulting averaged spectrum is highly complex in the range 1,000–4,000 kDa corresponding to the size of most conotoxins (10–30 amino acids). (c) Gel view representation of MALDI results reveals distinct regionalization of many venom components along the duct. For example, the predatory toxin at 3,175 kDa and defensive toxin at 1,417 kDa show clear non-overlapping distribution along the duct. (d) Quantification of five major predatory (including conopressin-G at 1,035 kDa) and defensive (including α-GII at 1,417 kDa, μ-GIIIA at 2,610 kDa and ω-GVIIA at 3,316 kDa) toxins confirms this region-specific toxin production. (e) Histology (formaldehyde-fixed animal embedded in paraffin) reveals structural heterogeneity along the venom duct, including regions with a dense layer of secretory cells and a small lumen and others with a looser cell arrangement and a larger lumen, which could support such regional specialization. Gomori’s Trichrome stain shows muscle fibres in red, collagen in green and nuclei in blue/black (scale bar, 20 μm). (f) A simple hypothesis to explain the generation of separate stimulus-evoked venoms is proposed. An initial stimulus (predatory or defensive) is perceived by mechanical, visual and/or chemical (olfactory) sensors that transmit information to the cerebral ganglia surrounding the oesophagus (O) to activate two separate neuronal circuits. Predation-evoked stimuli activate neuronal circuit (blue) innervating the distal venom duct, causing the release of predatory venom peptides into the venom duct lumen. Similarly, threats including larger fish and cephalopods activate a separate defensive neuronal circuit (green) that innervates the proximal venom duct, causing the release of defensive toxins into the lumen. These lumen contents are then moved to the proboscis by a synchronized contraction of the muscular venom bulb to generate the injected ‘predation-evoked’ and ‘defence-evoked’ venoms. This key role of the venom bulb allows the rapid switch between the predation- and defence-evoked venoms observed. This mechanism of stimulus-dependent release of toxins from different sections of the venom duct explains how distinct predation- and defence-evoked venoms are generated.

Figure 4. Molecular evolution of conotoxin gene superfamilies and the role of defensive evolutionary pressure on cone snail venom evolution.

(a) Molecular evolution assessment indicates that all C. geographus conotoxin superfamilies are rapidly evolving (ω>1). (b) Branch-site Random Effects Likelihood of superfamily O1 indicates the episodic nature of conotoxin evolution, revealing independent trajectories for defensive (green) or predatory (blue) conotoxins. (c) Specialization of the venom duct is a key evolutionary innovation in cone snails (venom duct and the associated proximal venom bulb are illustrated). We propose that the distinct defence (D) and predation-evoked (P) venoms found in the specialized venom ducts of modern cone snails evolved from an ancestral primitive cone snail that used the same venom produced in an unspecialized duct to deter predators and catch prey. This defensive behaviour, initially evolved to deter threats including cephalopod (octopus) and fish, likely triggered shifts in cone snail predatory strategies to mollusk- and fish-hunting that allowed predators to become prey.