Searching for synergism: effects of combinations of phenolic compounds and other toxins on oxidative stress in Lymantria dispar caterpillars

Synergistic combinations of plant allelochemicals are commonly believed to increase their effectiveness as defenses against insect herbivores. For example, temperate deciduous trees produce large amounts of phenolic compounds (primarily tannins), but many of these trees also produce smaller amounts of other potentially toxic compounds. This study tested the hypothesis that mixtures of phenolics and other types of toxins produce a greater effect on oxidative stress (a measure of toxicity) than do phenolics alone. Oxidative stress was measured in Lymantria dispar (gypsy moth) larvae as a shift in the redox balance of glutathione (GSH) towards a higher percentage of its oxidized form (glutathione disulfide; GSSG). We began by showing that larvae that ingested ellagitannins and chlorogenic acid (phenolics) contained greatly elevated levels of reactive oxygen species in their midgut contents, but this was not sufficient to shift the redox balance of GSH in their midgut tissues. Therefore, the phenolic compounds were tested in pairwise combinations with juglone (a quinone), rutin, kaempferol, or quercitrin (flavonoids), quinine, berberine, gramine, or glaucine (alkaloids), and soy or Quillaja saponins. When each of these allelochemical combinations was treated on hybrid poplar (Populus tremula × P. alba) leaves and consumed by L. dispar larvae for a 2-day period, none significantly shifted the redox balance of GSH. GSH levels were induced by the combination of phenolics with an alkaloid and a saponin, suggesting that a “cocktail” of allelochemicals might produce a more effective plant defense. GSH levels were also found to increase with larval age, both within and between instars. Increased levels of GSH may help explain the greater resistance of late-instar than early-instar larvae to plant defenses. Overall, the results of this study do not support the hypothesis that the combined effects of phenolic compounds and other toxins in sugar maple (Acer saccharum) leaves produce a synergistic increase in oxidative stress in L. dispar larvae. Further work is needed to test the general hypothesis that the allelochemical diversity in plants functions, in part, to produce synergistic toxicological effects in insect herbivores.