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

Abstract

array 27 28 29 Summary statement: 30 Analysis of key snail egg proteins shows evolutionary defensive trends associated with the 31 phylogenetic position, extending by convergent evolution the presence of plant-like defensive 32 strategies not reported in other animals 33 34 35 Abstract 36 The acquisition of egg protection is vital for species survival. Poisonous eggs from Pomacea 37 apple snails have defensive macromolecules for protection. Here we isolated and characterized 38 a novel lectin called PdPV1 that is massively accumulated in the eggs of Pomacea diffusa and 39 seems part of its protective cocktail. The native protein, an oligomer of ca. 256 kDa, has high 40 structural stability, withstanding 15 min boiling and denaturing by sodium dodecyl sulphate. It 41 resists in vitro proteinase digestion and displays structural stability between pH 2.0-12.0 and 42 up to 85 °C. These properties, as well as its subunit sequences, glycosylation pattern, presence 43 of carotenoids, size, and global shape resemble those of its orthologs from other Pomacea. 44 Further, like members of the canaliculata clade, PdPV1 is recovered unchanged in faeces of 45 mice ingesting it, supporting an antinutritive defensive function. PdPV1 also displays a strong 46 hemagglutinating activity specifically recognizing selected ganglioside motifs with high 47 affinity. This activity is only shared with PsSC, a perivitelline from the same clade (bridgesii 48 clade). As a whole, these results indicate that species in the genus Pomacea have diversified 49 their eggs defences: Those from the bridgesii clade are protected mostly by non-digestible 50 lectins that lower the nutritional value of eggs, in contrast with protection by neurotoxins of 51 other Pomacea clades, indicating apple snail egg defensive strategies are clade-specific. The 52 harsh gastrointestinal environment of predators would have favoured their appearance, 53 extending by convergent evolution the presence of plant-like highly stable lectins, a strategy 54 not reported in other animals. 55 98 structure, structural stability, and functional features in a phylogenetic framework. We suggest 99 a putative role of PdPV1 in embryo protection as both an antinutritive protein (highly stable, 100 nondigestible) and as a toxic lectin. Further, we describe an evolutionary association among 101 Pomacea egg defence systems, their phylogenetic position and invasiveness. 102 103 Methods 104 105 Ethics Statement 106 Studies performed with animals (Protocol Number: P01-01-2016) were approved by the 107