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

Apple snails (Pomacea Perry, 1810) are successful invaders, causing ecological perturbations, economic losses and medical issues. A peculiar trait of this snail is high biological potential, related to the absence of predators of their eggs. Eggs show protease inhibitor (PI) activity, originally ascribed to PcOvo perivitellin in Pomacea canaliculata (Lamarck, 1822) but absent in PmPV1, the orthologoue of PcOvo, in Pomacea maculata Perry, 1810 eggs. As egg fluid diminishes rat growth rate, an anti-digestive 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/proteomic sequencing and biochemical validation, we identified a Kunitz-type and a Kazal-type inhibitor which, 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.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author