Kelly S. Johnson

Plant Phenolics as Dietary Antioxidants for Herbivorous Insects: A Test with Genetically Modified Tobacco

High foliar phenolics are generally assumed to increase resistance to insect herbivores, but recent studies show that tobacco lines modified to over– and underexpress phenolics do not exhibit higher constitutive resistance to caterpillars. This is contrary to the expectation that ingestion of tobacco phenolics, particularly chlorogenic acid, should cause oxidative stress in herbivores. We investigated free radical production and antioxidant capacity of fresh crushed leaves of tobacco lines exhibiting over a sixfold difference in chlorogenic acid content to test whether high phenolic concentrations are associated with increased production of reactive oxygen species (ROS). The effects of in planta phenolic levels on feeding behavior, growth, biochemical markers of oxidative stress, and the antioxidant capacity of midgut fluid and hemolymph were assessed in tobacco budworm, Heliothis virescens. The experiments showed that high phenolic foliage was more prooxidant than low phenolic foliage, but the net balance in crushed tissue was antioxidant in comparison to buffer and the commercial antioxidant standard, Trolox. In H. virescens, the antioxidant capacity of midgut fluid was also powerful, and caterpillars fed high phenolic foliage did not exhibit the expected markers of oxidative stress in midgut tissues (altered ascorbate ratios, disulfides, or total hydroperoxides). Instead, hemolymph of larvae fed high phenolic foliage exhibited improved total Trolox equivalent antioxidant capacity (TEAC). These results suggest that the elevated foliar phenolics in some plants may have beneficial antioxidant properties for herbivorous insects, much as dietary phenolics do in mammals.

Oxygen levels in the gut lumens of herbivorous insects.

Oxygen levels were measured in the foregut and midgut lumens of ten species of caterpillars and three species of grasshoppers. In most species, the foregut was nearly anoxic, with oxygen levels ranging from 0 to 2.5 mm Hg. However, two caterpillar species with large foreguts (Malacosoma disstria and Lymantria dispar) had elevated oxygen levels (27.9 and 32.1 mm Hg) in this region when they were fed artificial diet. In all of the species surveyed, the anterior and posterior midgut were nearly anoxic, with oxygen levels ranging from 0 to 7.3 mm Hg. Oxygen levels in the midgut lumen of Helicoverpa zea did not differ when caterpillars were fed artificial diet or tomato foliage, suggesting that the insect is capable of reducing the level of ingested oxygen in its gut. An examination of the radial microgradient of oxygen in the gut lumen demonstrated that the midgut epithelium is not a sink for ingested oxygen. However, the midgut contents of larvae fed artificial diet were capable of depleting oxygen. This capacity was reduced by boiling, suggesting that the nearly anoxic state of the midgut lumen in some insects is maintained by endogenous chemical processes. We conclude that low oxygen levels in the gut lumens of most herbivorous insects may greatly reduce the rates of oxidation of ingested plant compounds by oxygen-dependent reactions.

Potential influence of midgut pH and redox potential on protein utilization in insect herbivores

Early insect physiologists recognized the importance of gut physicochemistry, primarily pH, redox potential, and ionic strength, on digestive processes, but studies to date have been primarily restricted to keratin- and wood-feeding insects. Recent investigations show that herbivorous insects have a broad range of gut redox conditions, with pHs ranging from 6.0 to 11.8 and measured redox potentials from −200 to +240 mV. The redox state of the gut is largely dependent on pH, which is well regulated, and the redox activity of ingested material, including plant chemicals, at that pH. Inter- and intraspecific variation in midgut redox conditions appears to be substantial enough to affect digestion via effects on the structure and function of dietary proteins and proteolytic enzymes. The impact of reducing conditions on proteins probably depends on characteristics such as tertiary structure and the number and arrangement of disulfide linkages. In addition to the effects of reducing conditions in dietary proteins, there can be effects on the activities of digestive enzymes, depending on their structure and the nature of their catalytic site. We speculate that phylogenetic and environmental determinants of gut physicochemistry may place constraints on the efficacy of different digestive processes, and may thus influence the evolution of digestive strategies in insects.

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.

Physiological and dietary influences on midgut redox conditions in generalist lepidopteran larvae

Abstract We examined the effects of host plant foliage and redox active compounds on the midgut physicochemistry of several species of lepidopteran larvae. Midgut pH varied among Helicoverpa zea, Heliothis virescens, Pseudoplusia includens and Hyphantria cunea but was not affected by the host plant they fed upon (cotton, clover, wild geranium or soybean). However, redox potential was influenced by both plant and lepidopteran species. Midgut redox potentials closely matched the redox potential of foliage homogenized under alkaline, but not neutral pH, suggesting that alkalinity has a strong influence on the reducing strength of foliage in the gut lumen. Artificial diet containing redox active compounds, including thiol reducing agents and plant allelochemicals, significantly lowered midgut redox potential of H. zea when ingested. Juglone induced a 205% drop in redox potential in the lumen, whereas caffeic acid, chlorogenic acid, pyruvate, cysteine, dithiothreitol and ascorbic acid had more moderate effects (34–44% reduction). Quebracho, tannic acid, o -coumaric and p -coumaric acids had no significant effects. These findings suggest that much of the variation in midgut redox conditions observed in folivorous caterpillars is due to the redox activity of host plant chemicals at the alkaline pH of the gut.

Digestive proteinase activity in corn earworm (Helicoverpa zea) after molting and in response to lowered redox potential.

Insect digestive proteinases are often strongly influenced by ambient physicochemical conditions, such as pH, ionic strength, and oxidation-reduction potential. Although the effects of the former two parameters are well documented, the influence of redox potential on catalytic rates of digestive enzymes is not well understood. In this study, we manipulated the midgut redox potential of a generalist caterpillar (the corn earworm, Helicoverpa zea) by augmenting artificial diet with dithiothreitol, a powerful thiol reducing agent that lowers the redox potential in the lumen by 40-45 mV. Effects on total proteolytic activity, as well as on elastase, chymotrypsin, trypsin, leucine aminopeptidase, and carboxypeptidase A and B activities were measured using azocasein and nitroanilide model substrates. The profiles of proteinase activities in the epithelium and lumen were also monitored on days 1, 2, and 3 after the molt in penultimate instar larvae. Although the reducing agent strongly inhibited the activity of some proteinases in vitro, ingestion of the reducing diet failed to affect in vivo proteinase activities. There was also no effect on larval relative growth, consumption, or digestive efficiencies. We conclude that dietary reducing agents must lower midgut redox potential to below -40 mV to significantly impact digestive efficiency. Arch.

Temporal Modulation of Pyrrolizidine Alkaloid Intake and Genetic Variation in Performance of Utetheisa ornatrix Caterpillars

Foraging on a variety of different food items allows individuals to balance nutrient intake, but it also may dilute toxins present in single items of the diet. The distinction between nutrient balancing and toxin dilution becomes less clear, however, for organisms that sequester potentially toxic compounds for their own benefit. Additionally, specific dietary components chosen may or may not affect metabolic rate, growth, and other measures of fitness. Using the arctiid moth Utetheisa ornatrix, we (1) evaluated the temporal pattern of larval feeding behavior when presented with diet enriched with the pyrrolizidine alkaloid (PA), monocrotaline, (2) estimated the cost of ingesting PAs by measuring larval nutritional performance on diets containing different amounts of usaramine, and (3) investigated the magnitude of genetic effects on the performance of families raised on diets of different usaramine concentrations. We demonstrate that an herbivorous insect can temporally vary its intake of sequestered allelochemical. Individuals combined a preference for intermediate amounts of monocrotaline with temporal modulation of the specific amount of PA ingested on a given day. Using usaramine, we found little evidence for a cost of ingesting PAs, except among some family groups. Measures of digestive and metabolic efficiency showed no consistent reduction in performance on higher alkaloid diets. Only when larvae in family groups were fed a single concentration of PA over more than one instar was there any evidence for a cost of ingestion. Additionally, there were large genetic components to both growth and an individuals growth response to dietary alkaloid. These results suggest that while genetic variation in performance on alkaloid-rich diets exists, modulation of intake rate by caterpillars may be an important mechanism to reduce effects on the growth and fitness of individuals.

Predicting mayfly recovery in acid mine-impaired streams using logistic regression models of in-stream habitat and water chemistry

Mayflies (Order Ephemeroptera) require high quality water and habitat in streams to thrive, so their appearance after restoration is an indicator of ecological recovery. To better understand the importance of restoring in-stream habitat versus water chemistry for macroinvertebrate communities, we developed taxon-specific models of occurrence for five mayfly genera (Caenis, Isonychia, Stenonema, Stenacron, and Baetis) inhabiting streams in the Appalachian Mountains, USA. Presence/absence records from past decades were used to develop single and multiple logistic predictive models based on catchment characteristics (drainage area, gradient), in-stream habitat variables (e.g., substrate, channel morphology, pool and riffle quality), and water chemistry. Model performance was evaluated using (a) classification rates and Hosmer-Lemeshow values for test sets of data withheld from the original model-building dataset and (b) a field comparison of predicted versus observed mayfly occurrences at 53 sites in acid mine drainage-impaired watersheds in 2012. The classification accuracies of final models for Caenis, Stenacron, and Baetis ranged from 50 to 75%. In-stream habitat features were not significant predictor variables for these three taxa, only water chemistry. Models for Isonychia and Stenonema had higher classification rates (81%) and included both habitat and chemical variables. However, actual occurrences of Isonychia and Stenonema at study sites in 2012 were low, consistent with the calculated probability of occurrence (Po) < 0.60. Caenis occurred at test sites 35% of the time when the model predicted a Po > 0.40. Stenacron showed the greatest consistency of actual versus predicted occurrences, occurring at 56% of sites when the Po (based on pH and conductivity) was > 0.50 and only at 1 site when Po < 0.5. The results demonstrate how predictive models of individual indicator taxa could be valuable for evaluating the relative impacts of restoring physical habitat versus water chemistry during stream remediation.