The Chlorophyll Catabolite, Pheophorbide a, 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, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, 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 m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 μg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 μg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 μg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 μg added Pha were comparable to intact shields, even though the Pha 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.

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

“…It was proposed that these proteins utilize bound chlorophyllide for antibacterial and antiviral activities of the larva (Pandian et al, 2008). Interestingly, tortoise beetle larvae have evolved to utilize pheophorbide a as a deterrent in a fecal shield to protect themselves from their predators (Vencl et al, 2009). A defensive compound could have contrasting effects against different insect species.…”

“…It is proposed that organisms use the toxicity of tetrapyrroles for their defense systems. The larvae of tortoise beetle (Chelymorpha alternans) even utilize pheophorbide a as a powerful deterrent in the fecal shield to protect themselves from their predators (Vencl et al, 2009). Kariola et al (2005) suggested that a chlorophyll derivative, chlorophyllide, is involved in the defense against fungi, based on their observations that down-regulation of a chlorophyllhydrolyzing enzyme, chlorophyllase (CLH), results in increased susceptibility of Arabidopsis (Arabidopsis thaliana) plants to the necrotrophic fungus Alternaria brassicicola.…”

Functional interactions among tortoise beetle larval defenses reveal trait suites and escalation

“…Because they both feed on the same host plant, Merremia umbellata (Convolvulaceae), their larvae confront the same enemies as they graze openly on leaves (e.g., Windsor et al 1992;Cuignet et al 2008). The larvae of both species have an unpalatable defense in the form of a physical and chemical barrier, or shield, which is mobile and fortified with host-derived chemicals (Vencl et al 2005(Vencl et al , 2009). Only A. sparsa employs a maternal care defense tactic, wherein females guard their offspring from egg until adult emergence (Windsor 1987).…”

An endophyte‐rich diet increases ant predation on a specialist herbivorous insect

“…Alternatively, endophytes may have made A. sparsa larvae less repellent by interfering with their production of defensive chemical compounds. The closely related tortoise beetle Chelymorpha alternans (Boheman) metabolises M. umbellata-derived chlorophyll in its gut to the catabolite pheophorbide a, which has been shown to deter A. lacrymosa ants in bioassays similar to those presented here (Vencl et al, 2009). Perhaps secondary metabolites particular to, or elevated in, E high leaves (Petrini et al, 1992) reduced the ability of the beetle larvae to convert chlorophyll to defensive chemical compounds, resulting in higher ant predation without necessarily having an effect on herbivore performance.…”

“…Trials began when an ant first antennated the larva, and were separated by 3 min to maximise independence by increasing the likelihood that different ants would be tested in each trial. We baited the platform with tuna 1 h prior to the first trial to ensure colony feeding motivation (Vencl et al, 2009); the ants' response to the bait and the thawed larvae was generally similar.…”