Rationale: Proteins from the egg perivitelline fluid (PVF) are assumed to play critical roles in embryonic development, but for many groups of animals their identities remain unknown. Identifying egg PVF proteins is a critical step towards understanding their functions including their roles in evolutionary transition in habitats. Methods: We applied proteomic and transcriptomic analysis to investigate the PVF proteome of the eggs of Pomacea diffusa, an aerial ovipositing freshwater snail in the family Ampullariidae. The PVF proteins were separated with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and proteomic analysis was conducted using an LTQ Velos ion trap mass spectrometer coupled with liquid chromatography. Comparison of PVF proteomes and evolution analyses was performed between P. diffusa and other ampullariids. Results In total, 32 egg PVF proteins were identified from P. diffusa. They were categorized as PV1-like subunits, immune-responsive proteins, protein degradation, signaling and binding, transcription and translation, metabolism, oxidation-reduction and proteins with unknown function. Interestingly, the proteome includes a calcium-binding protein important in forming the hard eggshell that enabled the terrestrial transition. However, it does not include PV2, a neurotoxic protein that was assumed to be present in all Pomacea species. Conclusions: The PVF proteome data from P. diffusa can help us better understand the roles that reproductive proteins played during the transition from underwater to terrestrial egg deposition. Moreover, they could be useful in comparative studies of the terrestrialization in several groups of animals that occurred independently during their evolution.Material suplementario: - Table S1.Results of P. diffusa PVF proteome using Mascot. - Table S2.Gene ontology (GO) annotation of PVF proteins in Pomacea diffusa. Gene ontology functions were annotated with InterProScan 5 and I-TASSER/COFACTOR (in bold). F: molecular function; P: biological process; C: cellular component. - Table S3.List of PVF protein coding gene orthogroup (proteins with two or more homologs) and singleton (unique protein without any homolog) genes. - Table S4.Results of the branch-site model test for detection of positively selected sites in genes with branch label in Figure S1. - Figure S1.Global ω values for each branch of Clade B. (A) calcium binding protein, (B) MACPF-like genes and (C) tachylectin-like genes inferred by the free-ratio model of Codeml. Branches with ω > 1 are indicated by red color. Green arrow indicates the branch with high albumen gland expression and selected for the branch-site test. - Figure S2.Alignment of (A) calcium binding protein, (B) MACPF-like and (C) tachylectin-like genes used in positive selection analysis. P. diffusa sequences are indicated by yellow arrows. Conserved residues are shaded in black and similar residues are in light gray. The sites with PP > 0.95 listed in Table S3 are highlighted in red box. - Figure S3.Protein domain of MAPCF (Pca_1306_1.39) by PROSITE. Positive selected site indicates with red arrow.Instituto de Investigaciones Bioquímicas de La Plata