Little is known about venom in young developmental stages of animals. The appearance of toxins and stinging cells during early embryonic stages in the sea anemone Nematostella vectensis suggests that venom is already expressed in eggs and larvae of this species. Here, we harness transcriptomic, biochemical and transgenic tools to study venom production dynamics in Nematostella. We find that venom composition and arsenal of toxin-producing cells change dramatically between developmental stages of this species. These findings can be explained by the vastly different interspecific interactions of each life stage, as individuals develop from a miniature non-feeding mobile planula to a larger sessile polyp that predates on other animals and interact differently with predators. Indeed, behavioral assays involving prey, predators and Nematostella are consistent with this hypothesis. Further, the results of this work suggest a much wider and dynamic venom landscape than initially appreciated in animals with a complex life cycle.

Highlights d Ctenophore neurons and sensory cells bear multiple unique neuropeptides d Several neuropeptides affect ctenophore swimming behavior d Peptidergic ctenophore neurons have long anastomosed neurites d Ctenophore neurons have both common and unique molecular and morphological features

The cnidarian Nematostella vectensis has become an established lab model, providing unique opportunities for venom evolution research. The Nematostella venom system is multimodal: involving both nematocytes and ectodermal gland cells, which produce a toxin mixture whose composition changes throughout the life cycle. Additionally, their modes of interaction with predators and prey vary between eggs, larvae, and adults, which is likely shaped by the dynamics of the venom system. Nv1 is a major component of adult venom, with activity against arthropods (through specific inhibition of sodium channel inactivation) and fish. Nv1 is encoded by a cluster of at least 12 nearly identical genes that were proposed to be undergoing concerted evolution. Surprisingly, we found that Nematostella venom includes several Nv1 paralogs escaping a pattern of general concerted evolution, despite belonging to the Nv1-like family. Here, we show two of these new toxins, Nv4 and Nv5, are lethal for zebrafish larvae but harmless to arthropods, unlike Nv1. Furthermore, unlike Nv1, the newly identified toxins are expressed in early life stages. Using transgenesis and immunostaining, we demonstrate that Nv4 and Nv5 are localized to ectodermal gland cells in larvae. The evolution of Nv4 and Nv5 can be described either as neofunctionalization or as subfunctionalization. Additionally, the Nv1-like family includes several pseudogenes being an example of nonfunctionalization and venom evolution through birth-and-death mechanism. Our findings reveal the evolutionary history for a toxin radiation and point toward the ecological function of the novel toxins constituting a complex cnidarian venom.

Venom is a complex trait with substantial inter- and intraspecific variability resulting from strong selective pressures acting on the expression of many toxic proteins. However, understanding the processes underlying toxin expression dynamics that determine the venom phenotype remains unresolved. By interspecific comparisons we reveal that toxin expression in sea anemones evolves rapidly and that in each species different toxin family dictates the venom phenotype by massive gene duplication events. In-depth analysis of the sea anemone, Nematostella vectensis, revealed striking variation of the dominant toxin (Nv1) diploid copy number across populations (1-24 copies) resulting from independent expansion/contraction events, which generate distinct haplotypes. Nv1 copy number correlates with expression at both the transcript and protein levels with one population having a near-complete loss of Nv1 production. Finally, we establish the dominant toxin hypothesis which incorporates observations in other venomous lineages that animals have convergently evolved a similar strategy in shaping their venom.