Non-digestible proteins and protease inhibitors: Implications for defense of the colored eggs of the freshwater apple snail pomacea canaliculata

Apple snails (Pomacea Perry, 1810) are successful invaders that cause ecological perturbations, economic losses, and medical issues. A peculiar trait of this snail is a high biological potential, related to the absence of predators of their eggs. Eggs show protease inhibitor (PI) activity, originally ascribed to PcOvo perivitellin in the apple snail Pomacea canaliculata (Lamarck, 1822) but absent in PmPV1, the orthologoue of PcOvo, in eggs of the apple snail Pomacea maculata Perry, 1810. As egg fluid diminishes rat growth rate, an antidigestive effect, similar to plant defenses against herbivory, was hypothesized. However, PI activity has not been characterized in apple snail eggs. Here we identify and partially characterize P. canaliculata egg PI and improve our knowledge of the quaternary structure and evolution of PcOvo. Through N-terminal, transcriptomic or proteomic sequencing, and biochemical validation, we identified a Kunitz-type and a Kazal-type inhibitor that, though at low concentration in the egg, exhibit strong PI activity against trypsin, chymotrypsin, elastase, and subtilisin. Additionally, we report three new subunits for the non-digestible storage protein PcOvo. They are likely products of ancient gene duplication, as their sequences exhibit moderate similarity (30%). To our knowledge, this is the first report of Kazal-type inhibition among invertebrate eggs. Inhibiting varied proteases, PI seems an efficient adaptive trait that limits predator’s capacity to digest egg nutrients.