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.