Alternative splicing serves as a molecular strategy for saliva evolution and diversification in an endoparasitoid, Pteromalus puparum

Animal saliva represents a powerful model for investigating adaptive evolution. In parasitoid wasps, salivary proteins are known to modulate host cellular and humoral immunity. However, the functional significance of widespread alternative mRNA isoforms derived from salivary genes remains largely unexplored. To fill this gap, we applied an integrative full-length isoform sequencing and profiling pipeline in the endoparasitoid wasp Pteromalus puparum, enabling the reconstruction of a high-resolution transcriptomic landscape of salivary genes. A total of 133 high-confidence salivary genes were identified, more than 75% of which produce multiple transcript isoforms. Mass spectrometry analysis suggested that alternative splicing contributes to salivary proteome diversity, with eight genes encoding distinct protein isoforms. Notably, 12 salivary genes displayed differential isoform usage with elevated expression in salivary glands relative to the carcass. A striking example is P. puparum serpin3 ( PpSerpin3 ), whose short isoform is specifically expressed in both salivary and venom glands. Functional assays revealed that this short isoform actively suppresses host humoral melanization. Given that active components in injected venom may gradually lose their efficacy, we propose that salivary secretions function to sustain host manipulation throughout parasitization. Comparative multi-omics analyses further showed that although salivary and venom systems share a conserved core genetic toolkit, they achieve functional specialization via tissue-specific gene family co-option and extensive isoform switching. This study presents an isoform-resolved transcriptomic framework of the parasitoid salivary system. The findings indicate that alternative splicing contributes to salivary protein diversity by encoding distinct protein products and, further, facilitates salivary gene evolution through gland-specific isoform switching. Collectively, this work provides mechanistic insights into the diversity and evolution of salivary systems.