Non-digestible proteins and protease inhibitors: implications for defense of the colored eggs of the freshwater apple snailPomacea canaliculata

Abstract

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.

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition

“…Among the orthogroups, 13 were found in all of the four ampullariids, including five PV1 subunits, five immune‐responsive proteins (C1q domain‐containing protein, CD109 antigen, deleted in malignant brain tumors 1, tachylectin‐related protein and soma ferritin), and three other proteins (superoxide dismutase 1, apoptosis‐inducing factor and novel protein). Interestingly, two orthogroups, CaBP that is involved in forming the egg shell, and Kunitz/Bovine pancreatic trypsin inhibitor that inhibits the functioning of multiple digestive proteases, were only detected in the three species of Pomacea. The presence of multiple immunoproteins in the PVF of the four species may indicate their role in defense against pathogens, as has been suggested for the eggs of the pulmonate snail Biomphalaria glabrata .…”

“…Nevertheless, P. diffusa and M. cornuarietis invest more on immune proteins and protease inhibitors. The three Pomacea species have a unique CaBP required for the formation of the calcareous shell of terrestrial eggs, and a Kunitz/Bovine pancreatic trypsin inhibitor that, at least in P. canaliculata and P. maculate , inhibit multiple digestive proteases of mice . In addition, duplication and positive selection of the CaBP and PV2 subunits were likely responsible for the aerial egg deposition and the evolution of the PV2 toxic protein as an anti‐predation mechanism in P. canaliculata and P. maculata .…”

“…43 multiple digestive proteases of mice. 7,36 In addition, duplication and positive selection of the CaBP and PV2 subunits were likely responsible for the aerial egg deposition and the evolution of the PV2 toxic protein as an anti-predation mechanism in P. canaliculata and P. maculata. 16 Overall, the results of this study will facilitate further research on the evolution of PVF proteins during the transition from underwater to terrestrial egg deposition in Ampullariidae, in particular the Old World lineage (e.g., the underwater ovipositor Lanistes and terrestrial ovipositor Pila).…”

A highly stable, nondigestible lectin from Pomacea diffusa unveils clade-related protection systems in apple snail eggs

“…PdPV1 is composed by six polypeptide chains similar to its orthologs PcOvo, PsSC and PmPV1 (Pasquevich et al, 2017;Ituarte et al, 2018Ituarte et al, , 2019 with only moderate sequence similarities among them. It has been suggested that they were generated by an ancient gene duplication (Sun et al, 2012).…”

A highly stable, nondigestible lectin fromPomacea diffusaunveils clade-related protection systems in apple snail eggs

“…PdPV1 is composed by six polypeptide chains similar to its orthologs PcOvo, PsSC and PmPV1 (Pasquevich et al ., 2017; Ituarte et al ., 2018, 2019) with only moderate sequence similarities among them. It has been suggested that they were generated by an ancient gene duplication (Sun et al ., 2012).…”