Stable yet Shifting: Early Toxin Dynamics in Typical and Atypical Clownfish–Anemone Symbioses

Among venomous animals, cnidarians represent the oldest metazoan lineage in which venom production and a specialized delivery system are defining synapomorphies. Cnidarians also represent the only venomous lineage for which mutualistic symbioses have evolved resulting in scenarios where mutualistic symbionts may also be targets of their venom. The most iconic example of this relationship is the mutualism between clownfish and their venomous sea anemone hosts. To investigate how symbiont presence and establishment influence toxin gene expression, we used a comparative TagSeq and RNA-Seq approach to quantify venom gene dynamics during the first 48 hours of clownfish–anemone symbiosis establishment in five anemone species. Our taxonomic sampling included three typical hosting species ( Entacmaea quadricolor , Radianthus crispa , and Stichodactyla haddoni ), each representing distinct evolutionary lineages of clownfish hosts, and two atypical Caribbean species ( Condylactis gigantea and Stichodactyla helianthus ) that do not host clownfish in nature, but have reported to host within the aquarium trade. Tentacle samples were collected prior to hosting, approximately 12 hours after initial symbiont establishment, and again 48 hours after symbiosis establishment. Our analyses revealed that overall toxin assemblages remained relatively stable during the early establishment phase, with no significant changes in the most highly expressed toxin gene candidates. However, subtle transcript-level shifts occurred within multi-copy toxin gene families, including cytolytic actinoporins and Sea Anemone 8 (SA8)-like toxins. Notably, one C. gigantea actinoporin transcript exhibited a ∼600-fold increase in expression in a single individual, which coincided with two clownfish mortalities prior to successful association, which subsequently decreased after establishment. Comparative sequence alignments suggest that amino acid substitutions in this transcript may be functionally relevant to symbiosis intolerance, as the amino acid substitutions were unique to this transcript, and not found in any other previously described cytolytic actinoporin. Together, these findings reveal that early toxin gene expression in clownfish-hosting sea anemones is largely stable, yet subtly dynamic at the transcript level. This study provides the first comparative transcriptomic insights into the molecular processes shaping symbiosis establishment in clownfish–anemone mutualisms, offering a framework for understanding venom evolution in the context of co-evolutionary interactions.