Sequestration of host plant carotenoids in the larval tissues of Helicoverpa zea.

To determine the cause of the unique yellow coloration in mandibular glands of soybean-fed Helicoverpa zea larvae, the accumulation of carotenoids in various tissues of last instar larvae fed soybean, cotton and tomato foliage was quantified. Five carotenoids were detected in the foliage of all host plants but at significantly different concentrations. Xanthophylls rather than carotenes were most likely to accumulate in larval tissues. Carotenoids accumulated at different rates and some were significantly affected by larval diet. Highest levels of carotenoid accumulation, notably lutein, were detected in the testes, followed by midgut epithelium, fat body and integument. The midgut epithelium contained the greatest and the testes the least diversity of carotenoid types. Low levels of lutein were detected in both labial and mandibular glands. Tomato foliage had the highest carotenoid content and caterpillar tissues fed these leaves often had the highest amounts of carotenoid. However, the accumulation of carotenoids did not protect larvae from antibiotic effects of tomato foliage because these caterpillars had the highest mortality and slowest growth rates of all the three host plants. Transport and absorption of lipid and oxidative stress may be some reasons for differential carotenoid accumulation.

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus.

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.

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.

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.

Trade-offs between pathogen and herbivore resistance.

During the past year genetic and pharmacological experiments have revealed a molecular basis for the cross-talk between signaling pathways mediating pathogen and herbivore resistance. These findings provide considerable insight into the apparently contradictory results reported for trade-offs between pathogen and herbivore resistance.

Communication between plants: induced resistance in wild tobacco plants following clipping of neighboring sagebrush.

The possibility of communication between plants was proposed nearly 20 years ago, although previous demonstrations have suffered from methodological problems and have not been widely accepted. Here we report the first rigorous, experimental evidence demonstrating that undamaged plants respond to cues released by neighbors to induce higher levels of resistance against herbivores in nature. Sagebrush plants that were clipped in the field released a pulse of an epimer of methyl jasmonate that has been shown to be a volatile signal capable of inducing resistance in wild tobacco. Wild tobacco plants with clipped sagebrush neighbors had increased levels of the putative defensive oxidative enzyme, polyphenol oxidase, relative to control tobacco plants with unclipped sagebrush neighbors. Tobacco plants near clipped sagebrush experienced greatly reduced levels of leaf damage by grasshoppers and cutworms during three field seasons compared to unclipped controls. This result was not caused by an altered light regime experienced by tobacco near clipped neighbors. Barriers to soil contact between tobacco and sagebrush did not reduce the difference in leaf damage although barriers that blocked air contact negated the effect.

Cross-talk between the signal pathways for pathogen-induced systemic acquired resistance and grazing-induced insect resistance.

Reducing phenylpropanoid biosynthesis in transgenic tobacco compromises systemic acquired resistance (SAR) to tobacco mosaic virus, while increasing phenylpropanoid biosynthesis enhances SAR. Surprisingly, transgenic tobacco plants compromised in SAR exhibit more effective grazing-induced systemic resistance to larvae of Heliothis virescens, whereas induced insect resistance is compromised in transgenic plants with elevated phenylpropanoid levels. Levels of the phenylpropanoid-derived signal salicylic acid are directly correlated with overall phenylpropanoid biosynthesis in this series of transgenic plants. Moreover, while pathogen-induced SAR is almost completely compromised in salicylic acid-deficient plants expressing the bacterial nahG salicylate hydroxylase gene, these plants show enhanced grazing-induced insect resistance compared to wild-type. Hence, suppression of grazing-induced insect resistance is mediated at least in part by salicylic acid and likely reflects salicylic acid inhibition of the synthesis and action of the wound signal jasmonic acid. We propose that the dual functions of salicylic acid contribute to a signal poise which constrains constitutive expression of disease and insect resistance mechanisms, and reciprocally switches their selective activation.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Antioxidant systems in insects.

Insects possess a suite of antioxidant enzymes and small molecular weight antioxidants that may form a concatenated response to an onslaught of dietary and endogenously produced oxidants. Antioxidant enzymes such as superoxide dismutase, catalase, glutathione transferase, and glutathione reductase have been characterized in insects. Water-soluble and lipid-soluble antioxidants such as ascorbate, glutathione, tocopherols, and carotenoids have not been well studied in insects but may play very important antioxidant roles. Additionally, the peritrophic matrix and trehalose may possess important antioxidant functions in insects. The enzymatic recycling of ascorbate, first noted in green plants, may also exist in insects. A greater understanding of these antioxidant systems may provide greater understanding about the ecological relationships of insects with their hosts.

Foliar oxidative stress and insect herbivory: Primary compounds, secondary metabolites, and reactive oxygen species as components of induced resistance.

Oxidative responses of plants to pathogens and other environmental stresses have received considerable recent attention. We propose that an oxidative response also occurs following attack by herbivores. Our data strongly indicate a shift in the oxidative status of soybean following herbivory by the insectHelicoverpa zea. Herbivory caused significant increases in lipid peroxidation and ·OH radical formation. The activity of several oxidative enzymes including lipoxygenases, peroxidase, diamine oxidase, ascorbate oxidase, and NADH oxidase I increased after herbivory on soybean. The enhanced production of phenolic compounds is indicated by an increase in the activity of phenylalanine ammonia lyase in wounded tissues. On the other hand, the level of soybean foliar antioxidants such as ascorbic acid, total carotenoids, nonprotein thiols, and catalase decreased significantly following herbivory. These results implicate primary compounds (e.g., ascorbic acid, proteins), secondary metabolites (e.g., phenolics), and reactive oxygen species (e.g., hydroxyl radical, hydrogen peroxide) as multiple components of induced resistance. The oxidative changes in the host plant correspond with increased oxidative damage in the midgut of insects feeding on previously wounded plants. Decreases in nonprotein thiols and reduced ascorbic acid occurred in midgut epithelial tissue from insects feeding on wounded plants compared to the insects on control plants. In contrast, midgut hydroperoxides and dehydroascorbic acid concentrations were greater in insects on wounded plants compared to their counterparts on control plants. We conclude that oxidative responses in soybean may have both positive and negative effects upon the host plant: a decrease in herbivory and an increase in oxidative damage to the plant. The salient benefit to the plant, in terms of insect resistance, is the relative balance between these opposing effects.

Induced resistance in soybean toHelicoverpa zea: Role of plant protein quality.

Resistance in soybean toHelicoverpa zea is comprised of both constitutive and inducible factors. In this study, we investigated the induction of resistance byH. zea in both greenhouse and field studies. In a greenhouse experiment, fourth-instarH. zea growth rates were reduced by 39% after 24 hr feeding and by 27% after 48 hr when larvae fed on previously wounded V3 foliage (cv. Forrest) compared with undamaged foliage. In a field study, the weight gain by larvae was more than 52% greater when larvae fed for 72 hr on undamaged R2/R3 soybean plants (cv. Braxton) compared to those that fed on previously wounded plants. A significant component of the induced resistance is due to a decline in the nutritional quality of foliar protein following foliar damage byH. zea. Foliar protein was extracted from damaged and undamaged foliage and incorporated into artificial diets. Larval growth was reduced 26% after four days and 49% after seven days on diets containing protein from damaged plants compared to larvae feeding on foliar protein from undamaged plants. Chemical analyses of protein quality also indicated a decline in quality in damaged plants compared to unwounded plants. Increases in lipoxygenase activity (53%), lipid peroxidation products (20%), and trypsin inhibitor content (34%) were observed in protein from wounded plants. Moreover, a 5.9% loss in free amines and 19% loss in total thiols occurred in protein from wounded plants. Larval feeding causes a significant increase in foliar lipoxygenase activity that varied among genotypes. Lipoxygenase isozymes were measured at pH 5.5, pH 7.0, and pH 8.5 in V3 stage plants of Forrest, Hark, D75-1069, and PI 417061 genotypes. Lipoxygenase activity in each genotype was significantly increased after 72 hr of larval feeding at each pH level tested, with the exception of lipoxygenase isozymes at pH 5.5 in genotype PI 417061. Larval feeding on R2/R3 stage plants (field-grown cv. Braxton) for six days also increased foliar lipoxygenase activity.

Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper.

Variation in induced responses in soybean is shown to be dependent, in part, upon herbivore species. Herbivory by the phloem-feeding three-cornered alfalfa hopper caused increases in the activities of several oxidative enzymes including lipoxygenases, peroxidases, ascorbate oxidase, and polyphenol oxidase. Bean leaf beetle defoliation caused increased lipoxygenase activity, but had little effect upon peroxidase, polyphenol oxidase, ascorbate oxidase, or trypsin inhibitor levels in either field or greenhouse studies. In one field experiment, prior herbivory by the bean leaf beetle subsequently reduced the suitability of foliage to the corn earwormHelicoverpa zea. The contribution of these findings to emerging theories of insect-plant interactions is discussed.

Potential role of lipoxygenases in defense against insect herbivory.

The potential role of the plant enzyme lipoxygenase in host resistance against the corn earwormHelicoverpa zea was examined. Lipoxygenase is present in most of the common host plants ofH. zea, with highest activity in the leguminous hosts such as soybean and redbean. Treatment of dietary proteins with linoleic acid and lipoxygenase significantly reduced the nutritive quality of soybean protein and soy foliar protein. Larval growth was reduced from 24 to 63% depending upon treatment. Feeding byH. zea on soybean plants caused damage-induced increases in foliar lipoxygenase and lipid peroxidation products. Larvae feeding on previously wounded plant tissue demonstrated decreased growth rates compared to larvae feeding on unwounded tissue. Midgut epithelium from larvae feeding on wounded tissues showed evidence of oxidative damage as indicated by significant increases in lipid peroxidation products and losses in free primary amines. The potential role of oxidative and nutritional stress as a plant defensive response to herbivory is discussed.

Antioxidant role of dehydroascorbic acid reductase in insects.

Dehydroascorbic acid reductase, which catalyses the regeneration of ascorbic acid from dehydroascorbic acid, is reported here to occur widely among insects. Due to the reported absence of glutathione peroxidase in insects and the generally low affinity of catalase for hydrogen peroxide, dehydroascorbic acid reductase may play a pivotal role in the elimination of hydrogen peroxide in insects.

Potential role of ascorbate oxidase as a plant defense protein against insect herbivory.

Ascorbic acid is essential for both nutritive and antioxidant functions in phytophagous insects; however, maintaining sufficient quantities of reduced ascorbate may be problematical for them. In this investigation, we show that the plant enzyme ascorbate oxidase retains activity in the digestive system of the herbivoreHelicoverpa zea. High levels of the enzyme are present in several host plants ofH. zea, including cotton, tomato, soybean, crimson clover, and vetch. The enzyme oxidizesL-ascorbic acid to dehydro-L-ascorbic acid, a potentially toxic product. The oxidation of ascorbic acid also produces active oxygen species such as the highly reactive hydroxyl radical. The nutritional quality of protein for larvalH. zea was significantly reduced by treatment with ascorbate and ascorbate oxidase. Oxidative damage to the protein was indicated by decreased lysine content, increased carbonyl formation, and the occurrence of protein fragmentation and polymerization. Furthermore, the oxidative loss of ascorbate in the herbivore's digestive system prevents ascorbate from functioning as an important antioxidant against a plethora of dietary prooxidants.

Avoidance of antinutritive plant defense: Role of midgut pH in Colorado potato beetle.

The fate of the tomato foliar phenolic, chlorogenic acid, in the digestive systems of Colorado potato beetleLeptinotarsa decemlineata (Coleoptera: Chrysomelidae) andHelicoverpa tea (Lepidoptera: Noctuidae) is compared. In larvalH. zea and other lepidopteran species previously examined, approximately 35-50% of the ingested chlorogenic acid was oxidized in the digestive system by foliar phenolic oxidases (i.e., polyphenol oxidase and peroxidase) from the tomato plant. The oxidized form of chlorogenic acid, chlorogenoquinone, is a potent alkylator of dietary protein and can exert a strong antinutritive effect upon larvae through chemical degradation of essential amino acids. In contrast, inL. decemlineata less than 4% of the ingested dose of chlorogenic acid was bound to protein. In vitro experiments to determine the influence of pH on covalent binding of chlorogenic acid to protein showed that 30-45% less chlorogenic acid bound to protein at pHs representative of the beetle midgut (pH 5.5-6.5) than at a pH representing the lepidopteran midgut (pH 8.5). At an acidic pH, considerably more of the alkylatable functional groups of amino acids (-NH2, -SH) are in the nonreactive, protonated state. Hence, polyphenol oxidases are unlikely to have significant antinutritive effects against the Colorado potato beetle and may not be a useful biochemical source of resistance against this insect. The influence of feeding by larval Colorado potato beetle on foliar polyphenol oxidase activity in tomato foliage and its possible significance to interspecific competition is also considered.

Protective action of midgut catalase in lepidopteran larvae against oxidative plant defenses.

Catalase activity was detected in the midgut tissues and regurgitate of several lepidopteran pests of the tomato plant. Greatest activity in the midgut was detected in larvalHelicoverpa zea, followed bySpodoptera exigua, Manduca sexta, andHeliothis virescens. We present evidence that catalase, in addition to removing toxic hydrogen peroxide, may inhibit the oxidation of plant phenolics mediated by plant peroxidases. Small amounts of larval regurgitate significantly inhibited foliar peroxidase activity via removal of hydrogen peroxide. Treatment of foliage with purified catalase nearly eliminated peroxidase activity and was superior as a larval food source compared to untreated foliage. Tomato foliar peroxidases oxidize an array of endogenous compounds including caffeic acid, chlorogenic acid, rutin, coumaric acid, cinnamic acid, and guaiacol. The oxidized forms of these compounds are potent alkylators of dietary and/or cellular nucleophiles (e.g., thiol and amino functions of proteins, peptides, and amines). When tomato foliar protein was pretreated with peroxidase and chlorogenic acid and incorporated in artificial diet, larval growth was reduced compared to larvae fed untreated protein. Thus, the diminution of peroxidase activity and removal of hydrogen peroxide by catalase may represent an important adaptation to leaf-feeding. The secretion of catalase in salivary fluid during insect feeding is also suggested to be a potential mechanism for reducing hydrogen peroxide formation as an elicitor of inducible plant defenses.

Reassessment of the role of gut alkalinity and detergency in insect herbivory.

Previously it was reported that significant amounts of the tomato phenolic, chlorogenic acid, were oxidized in the digestive system of generalist feedersSpodoplera exigua andHelicoverpa zea. The covalent binding of the oxidized phenolic (i.e., quinone) to dietary protein exerts a strong antinutritive effect against larvae. In this study, we examined the fate of ingested chlorogenic acid in larvalManduca sexta, a leaf-feeding specialist of solanaceous plants. Significant amounts of chlorogenic acid were bound to excreted protein byM. sexta when larvae fed on tomato foliage. However, in the case ofM. sexta we suggest that the strong alkalinity and detergency of the midgut may minimize the antinutritive effects of oxidized phenolics. The solubility of tomato leaf protein is significantly greater at pH 9.7, representative of the midgut ofM. sexta, than at pH 8.0, representative of the midguts ofH. zea and S. exigua. We suggest that this increase in solubility would compensate for any loss in bioavailability of essential amino acids caused by the covalent binding of chlorogenic acid to amino acids. Furthermore, lysolecithin, a surfactant likely to contribute to the detergent properties of the midgut fluid, was shown to enhance protein solubility as well as inhibit polyphenol oxidase activity. The adaptive significance of gut alkalinity and detergency is discussed.

Plant defenses: Chlorogenic acid and polyphenol oxidase enhance toxicity ofBacillus thuringiensis subsp.kurstaki toHeliothis zea.

Two chemicals implicated in resistance of the tomato plant, chlorogenic acid and polyphenol oxidase, are known to form orthoquinones in damaged plant tissue. Orthoquinones have been reported to alkylate -NH2 and -SH groups of proteins and amino acids, altering solubility, digestibility, and, for some pathogenic viruses, infectivity. Here we explore effects of quinone alkylation on toxicity of an important microbial insecticide,Bacillus thuringiensis subsp.kurstaki (BTk), to larvalHeliothis zea. BTk incubated with these phytochemicals and fed to larvae was more toxic than untreated BTk. Similar but less dramatic results arose when BTk was incubated with polyphenol oxidase alone. Digestibility experiments suggest that alkylation enhanced the solubilization and/or proteolysis of crystal protein in vivo. Implications of our results for compatibility of BTk with host-plant resistance and biological control are discussed.

Inactivation of baculovirus by quinones formed in insect-damaged plant tissues.

The infectivity of the nuclear polyhedrosis virus, HzSNPV toHeliothis zea was significantly reduced when viral occlusion bodies were exposed to the plant phenolic chlorogenic acid in the presence of polyphenol oxidase. Chlorogenic acid is rapidly oxidized to the ortho-quinone, chlorogenoquinone, by foliar polyphenol oxidases of the tomato plant, Lycopersicon esculentum, when foliage is damaged during feeding by larvalH. zea.Our results indicate that chlorogenoquinone, a powerful oxidizing agent, covalently binds to the occlusion bodies of HzSNPV and significantly reduces their digestibility and solubility under alkaline conditions. This binding is proposed to interfere with the infection process by impairing the release of infective virions in the midgut.