Two Defensive Lines in Juvenile Leaf Beetles; Esters of 3-nitropropionic Acid in the Hemolymph and Aposematic Warning

Juveniles of the leaf beetles in subtribe Chrysomelina have efficient defense strategies against predators. When disturbed, they transiently expose volatile deterrents in large droplets from nine pairs of defensive glands on their back. Here, we report on an additional line of defense consisting of the non-volatile isoxazolin-5-one glucoside and its 3-nitropropanoyl ester in the larval hemolymph. Because isoxazolin-5-one derivatives were not detectable in related leaf beetle taxa, they serve as a diagnostic marker for the Chrysomelina subtribe. Conjugation of isotopically labelled 3-nitropropionic acid to isoxazolin-5-one glucoside in vivo demonstrates its function as a carrier for the 3-nitropropanoyl esters. The previous identification of characteristic glucosides as precursors of the volatile deterrents underlines the general importance of glucosides for sequestration from food plants, and the subsequent transport in the hemolymph to the defense system. The combination of repellent volatiles with non-volatile toxic compounds in the hemolymph has the potential to create synergistic effects since the odorant stimulus may help predators learn to avoid some foods. The combination of the two defense lines has the advantage, that the hemolymph toxins provide reliable and durable protection, while the repellents may vary after a host plant change.