Neurons, Muscles, and Venom: Identifying Drivers of Cephalopod Predation

Venom plays a central role in the predatory ecology of coleoid cephalopods (octopuses, squids, and cuttlefish), yet the mechanisms governing venom release from the posterior salivary gland (PSG) are unknown. Using a multimodal approach combining histology, in situ hybridization, comparative phylogenetics, and live imaging across multiple coleoid species, we characterize the structural and neuronal regulatory organization of the PSG. We show that the gland comprises two distinct tubular systems: secretory tubules specialized for venom production and smooth-striated tubules positioned to facilitate venom transport toward the beak for injection into its prey. Molecular localization of filamentous and -actin confirms a circular smooth muscle layer surrounding the tubules. Mapping of six neuronal markers, including neurofilament (NF-H), synapsin, and muscle-type nicotinic acetylcholine receptors, reveals dense and stereotyped neural innervation closely associated with the muscular compartments. Comparative phylogenetic analyses of cys-loop ligand-gated ion channel sequences indicate a predominancy of excitatory acetylcholine- and dopamine-gated receptors in coleoid venom glands, implicating potential molecular agents involved in neural control of venom release. Consistent with neural regulation, ex vivo stimulation of the PSG elicits calcium signaling throughout the gland. Together, our results reveal a conserved venom gland structure among octopuses, squids, and cuttlefish, with a spatially distinct neuromuscular tissue organization indicating venom production and release sites modulated by a network of neuronal agents. This work provides a mechanistic framework into venom gland organization and molecular regulation of venom release in one of the oldest venomous lineages. Significance StatementColeoid cephalopods (octopuses, squids, and cuttlefish) are predominantly known for their camouflage abilities and less for their use of venom in prey capture, defense, and sexual competition. How cephalopods release their venoms remains an open question. Our results indicate that the venom gland is differentiated into venom production and release tubular sections This bifurcation is conserved across several taxa including the longfin inshore squid, the hummingbird bobtail squid, and the California two-spot octopus, with few lineage-specific variations. Spatial molecular mapping revealed smooth muscle components and cholinergic neurons in intimate association with gland tubules. Comparative phylogenetic analyses further enhance neuronal network understanding by transcriptionally identifying conserved nicotinic acetylcholine-gated channels, divergent dopamine-gated channels, and multiple orphan ligand-gated ion channels with potentially novel functional properties. Our findings provide a comprehensive characterization of the network of molecular and neuronal components that can act in concert to facilitate cephalopod venom release. Future expansion and refinement of these results will enable mapping of the brain-bite neural circuit underlying cephalopod predation.