Supplementary file 1_Multiple egg defenses arose early in the history of apple snails preceding carotenoid-bound perivitellins.pdf

Nonetheless, mass spectrometry revealed multiple PV1 subunits, representing the first evidence of a carotenoid-free PV1 in Pomacea. Despite lacking the dominant carotenoid-binding form of PV1, other components of the multilayered defense system were found in P. sordida eggs. Several proteins (22–66 kDa) were resistant to simulated gastrointestinal and fungal digestion, and PVF has strong protease inhibitors of animal, bacterial, and fungal origin (40-87% inhibition).

Additionally, lectin-like activity agglutinated erythrocytes and both Gram-positive and Gram-negative bacteria. PV2-like neuro-enterotoxin was detected, compatible with the finding that the egg extracts were lethal to mice when injected and, when administered orally, induced marked but reversible morphological alterations in the murine intestine, including reduced absorptive surface, villus edema, and epithelial degeneration.

Overall, these results demonstrate that key components of Pomacea egg-defense repertoire are already present in a basal lineage, suggesting that the evolution of the protein-based defense system observed in derived clades arose early in the history of the genus and later diversified into distinct clade-specific defensive strategies.</p>