Egg volatiles of oak silkworm mediate defense against parasitoid wasps

Most insects protect their eggs from natural enemies via physical coverings, yet defensive mechanisms of exposed eggs (i.e., those without such coverings), particularly those lacking toxins, remain largely unexplored. The oak silkworm (Antheraea pernyi) lays such exposed eggs, yet these eggs exhibit less parasitism than manually extracted washed eggs (MUWE) under laboratory conditions, raising the question of whether this defense operates in nature and how it functions. Here, field experiments showed that naturally laid unwashed eggs had significantly lower parasitism by three parasitoid species than MUWE. Behavioral parasitism assays showed the specialist M. trabalae nearly avoided intact naturally laid unwashed eggs (≤2.5%), while generalist A. japonicus displayed fewer contacts and oviposition attempts. Y-tube olfactometry verified egg-derived volatiles mediate this repellency. Gas chromatography-mass spectrometry analysis identified 2-ethyl-1-hexanol (2EH) as a key volatile unique to naturally laid eggs. Electroantennography confirmed dose-dependent antennal responses to 2EH in both species. Y-tube assays showed that 2EH repels A. japonicus at ≥ 5 µg and M. trabalae at doses as low as 500 ng. Crucially, supplementing MUWE with 2EH greatly decreased the parasitism success in both parasitoids. This study reveals that egg-associated 2EH acts as a chemical shield protecting exposed eggs from parasitoids, demonstrating that exposed eggs are not inherently vulnerable but rather employ volatile-mediated defense against natural enemies. These findings advance our understanding of insect egg defense strategies, illuminate the evolutionary arms race between hosts and parasitoids, and establish a theoretical foundation for optimizing natural enemy mass rearing and developing behavioral regulators in biological control.