The chlorophyll catabolite, pheophorbide <I>a</I>, confers predation resistance in a larval tortoise beetle shield defense

Abstract

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, <I>Chelymorpha alternans</I> (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with <I>C. alternans</I> feeding on <I>Merremia umbellata</I> (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, <I>Azteca lacrymosa</I>, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at <I>m</I>/<I>z</I> 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide <I>a</I> (Ph<I>a</I>) identity. <I>C. alternans</I> shields contained more than 100 µg Ph<I>a</I> per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 µg of Ph<I>a</I> were more deterrent than larvae with solvent-leached shields, while those with 5 µg additional Ph<I>a</I> provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 µg added Ph<I>a</I> were comparable to intact shields, even though the Ph<I>a</I> concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.