Sea Anemone Membrane Attack Complex/Perforin Superfamily Demonstrates an Evolutionary Transitional State between Venomous and Developmental Functions

Abstract

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.

Keywords: Cnidaria; gene duplication; reverse recruitment; subfunctionalization; toxin; venom.

Fig. 1.

Phylogeny of MAC genes across Anthozoa. Maximum-likelihood tree of the MACs in anthozoans. The sequences found in nematocytes appear in bold with a magenta/elliptical nematocyte cartoon. The nematocyte-specific expression is characterized using cell atlases from different anthozoans including Xenia sp., S. pistillata, E. diaphana, and Nematostella (Hu et al. 2020; Levy et al. 2021; Steger et al. 2022; Cui et al. 2023). Additional NveMACs are found to have nematocyte-specific expression using bulk RNA-seq of the Nematostella transgenic line expressing NvNcol3::memOrange2, a nematocyte marker (Sunagar et al. 2018; Gahan et al. 2022). Sequences found to have high levels of expression in the Endo-atlas of Nematostella (He et al. 2023) also contain a gray/circular cartoon representing the endomesodermal segments. MACs found in the venom and isolated from nematocytes of anthozoans are highlighted with an asterisk as well as in bold with a magenta nematocyte cartoon. Ultrafast bootstrap values and SH-like approximate likelihood ratio test above 85 are indicated. Taxon identifies can be found supplementary table S1, Supplementary Material online. Silhouettes were made from BioRender.com.

Fig. 2.

Schematic representation depicting the MAC gene cluster and gene synteny among Anthozoans. Dashed lines between N. vectensis and S. callimorphus represent a cluster that shares macrosynteny. Created with BioRender.com

Fig. 3.

Spatiotemporal expression of MAC-encoding genes in Nematostella. a) ISH of NveMACs (1 to 4) across embryonic development. b) Graph of RNA levels of the same MAC genes used in ISH. Scale bar represents 100 μm. c) PCA using temporal expression of NveMACs in Nematostella throughout embryonic development taken from the NvERTx database. UE, unfertilized egg; BL, blastula; GA, gastrula; EP, early planula; LP, late planula; MM, metamorphosis; PP, primary polyp; JP, juvenile polyp; AP, adult polyp.

Fig. 4.

Knockdown of MACs in Nematostella. qRT-PCR for NveMACs at late planula stage after shRNA injection. The graph shows the relative fold change in the expression between control shRNA and the combined knockdown of a) NveMAC1, b) NveMAC3, and c) NveMAC4, using sequence-specific shRNA. The values for the individual replicates are shown as circles. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. d) Quantification of normal polyp development following the injection of control shRNA or knockdown of NveMACs 1, 3, and 4 simultaneously in 10 dpf polyps. e) Control shRNA. f) NveMACs 1, 3, and 4 shRNA. *P-value < 0.05; NS, not significant; **P-value < 0.01; ***P-value < 0.001. Scale bar represents 200 μm.

Fig. 5.

Reconstructing evolutionary history and possible scenarios of the origin of NveMACS as either reverse recruitment (a) or subfunctionalization (b). Horizontal arrows represent duplication event. Nematocyte expression is represented as magenta boxes and nematocyte cartoon, endomesodermal expression is represented by gray boxes, and cartoon represents the endomesodermal segments. Expression in both cell types is represented by boxes with both pink and gray and cartoons depicting both nematocyte and endomesodermal segments.