Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin.

As a managed agricultural pollinator, the western honeybee Apis mellifera frequently encounters agrochemicals as contaminants of nectar and pollen. One such contaminant, the fungicide boscalid, is applied at bloom in orchards for fungal floral pathogen control. As an inhibitor of complex II in the mitochondrial electron transport chain of fungi, boscalid can potentially interfere with high energy-demanding activities of bees, including flight. We designed an indoor flight treadmill to evaluate impacts of ingesting boscalid and/or quercetin, a ubiquitous phytochemical in bee food that also affects mitochondrial respiration. Boscalid reduced the wingbeat frequencies of foragers during flight but did not alter the duration of flight. At the colony level, boscalid ingestion may thereby affect overall health by reducing forager efficiency. The consumption of quercetin, by contrast, led to higher adenosine triphosphate levels in flight muscles and a higher wingbeat frequency. Consuming the two compounds together increased wingbeat frequency, demonstrating a hitherto unrecognized mechanism by which dietary phytochemicals may act to ameliorate toxic effects of pesticides to promote honeybee health. In carrying out this work, we also introduce two methodological improvements for use in testing for pesticide effects on flight capacity-a 'force-feeding' to standardize flight fuel supply and a novel indoor flight treadmill.

Cytochrome P450 diversification and hostplant utilization patterns in specialist and generalist moths: Birth, death and adaptation.

Across insect genomes, the size of the cytochrome P450 monooxygenase (CYP) gene superfamily varies widely. CYPome size variation has been attributed to reciprocal adaptive radiations in insect detoxification genes in response to plant biosynthetic gene radiations driven by co-evolution between herbivores and their chemically defended hostplants. Alternatively, variation in CYPome size may be due to random "birth-and-death" processes, whereby exponential increase via gene duplications is limited by random decay via gene death or transition via divergence. We examined CYPome diversification in the genomes of seven Lepidoptera species varying in host breadth from monophagous (Bombyx mori) to highly polyphagous (Amyelois transitella). CYPome size largely reflects the size of Clan 3, the clan associated with xenobiotic detoxification, and to some extent phylogenetic age. Consistently across genomes, families CYP6, CYP9 and CYP321 are most diverse and CYP6AB, CYP6AE, CYP6B, CYP9A and CYP9G are most diverse among subfamilies. Higher gene number in subfamilies is due to duplications occurring primarily after speciation and specialization ("P450 blooms"), and the genes are arranged in clusters, indicative of active duplicating loci. In the parsnip webworm, Depressaria pastinacella, gene expression levels in large subfamilies are high relative to smaller subfamilies. Functional and phylogenetic data suggest a correlation between highly dynamic loci (reflective of extensive gene duplication, functionalization and in some cases loss) and the ability of enzymes encoded by these genes to metabolize hostplant defences, consistent with an adaptive, nonrandom process driven by ecological interactions.

Disruption of quercetin metabolism by fungicide affects energy production in honey bees (Apis mellifera).

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.

Impacts of Dietary Phytochemicals in the Presence and Absence of Pesticides on Longevity of Honey Bees (Apis mellifera).

Because certain flavonols and phenolic acids are found in pollen and nectar of most angiosperms, they are routinely ingested by Apis mellifera, the western honey bee. The flavonol quercetin and the phenolic acid p-coumaric acid are known to upregulate detoxification enzymes in adult bees; their presence or absence in the diet may thus affect the toxicity of ingested pesticides. We conducted a series of longevity assays with one-day-old adult workers to test if dietary phytochemicals enhance longevity and pesticide tolerance. One-day-old bees were maintained on sugar syrup with or without casein (a phytochemical-free protein source) in the presence or absence of quercetin and p-coumaric acid as well as in the presence or absence of two pyrethroid insecticides, bifenthrin and ß-cyfluthrin. Dietary quercetin (hazard ratio, HR = 0.82), p-coumaric acid (HR = 0.91) and casein (HR = 0.74) were associated with extended lifespan and the two pyrethroid insecticides, 4 ppm bifenthrin (HR = 9.17) and 0.5 ppm ß-cyfluthrin (HR = 1.34), reduced lifespan. Dietary quercetin enhanced tolerance of both pyrethroids; p-coumaric acid had a similar effect trend, although of reduced magnitude. Casein in the diet appears to eliminate the life-prolonging effect of p-coumaric acid in the absence of quercetin. Collectively, these assays demonstrate that dietary phytochemicals influence honey bee longevity and pesticide stress; substituting sugar syrups for honey or yeast/soy flour patties may thus have hitherto unrecognized impacts on adult bee health.

Impact of Pesticide Resistance on Toxicity and Tolerance of Hostplant Phytochemicals in Amyelois Transitella (Lepidoptera: Pyralidae).

For some polyphagous insects, adaptation to phytochemically novel plants can enhance resistance to certain pesticides, but whether pesticide resistance expands tolerance to phytochemicals has not been examined. Amyelois transitella Walker (navel orangeworm) is an important polyphagous pest of nut and fruit tree crops in California. Bifenthrin resistance, partially attributable to enhanced cytochrome P450 (P450)-mediated detoxification, has been reported in an almond-infesting population exposed to intense pesticide selection. We compared the toxicity of bifenthrin and three phytochemicals-chlorogenic acid, and the furanocoumarins xanthotoxin and bergapten-to three strains of A. transitella: pyrethroid-resistant R347 (maintained in the laboratory for ∼10 generations), fig-derived FIG (in the laboratory for ∼25 generations), and CPQ-a laboratory strain derived from almonds ∼40 years ago). Whereas both Ficus carica (fig) and Prunus dulcis (almond) contain chlorogenic acid, furanocoumarins occur only in figs. Both R347 and FIG exhibited 2-fold greater resistance to the three phytochemicals compared with CPQ; surprisingly, bifenthrin resistance was highest in FIG. Piperonyl butoxide, a P450 synergist, increased toxicity of all three phytochemicals only in CPQ, implicating alternate tolerance mechanisms in R347 and FIG. To test the ability of the strains to utilize novel hostplants directly, we compared survival on diets containing seeds of Wisteria sinensis and Prosopis pallida, two non-host Fabaceae species; survival of FIG was highest and survival of R347 was lowest. Our results suggest that, while P450-mediated pesticide resistance enhances tolerance of certain phytochemicals in this species, it is only one of multiple biochemical adaptations associated with acquiring novel hostplants.

Larval Preference and Performance of Amyelois transitella (Navel Orangeworm, Lepidoptera: Pyralidae) in Relation to the Fungus Aspergillus flavus.

The navel orangeworm, Amyelois transitella (Walker), is a polyphagous pest of California nut crops and is responsible for extensive losses in the United States. It directly damages crops by feeding and contaminating nuts with frass and webbing and vectors saprophytic fungi that infect crops. The navel orangeworm is commonly associated with Aspergillus species, including the toxigenic Aspergillus flavus, which causes crop loss by producing carcinogens, including aflatoxin B1. This lepidopteran-fungus association is the most economically serious pest complex in Central Valley orchards, and evidence indicates that this relationship is mutualistic. We assessed preference and performance of navel orangeworm larvae associated with A. flavus in behavioral bioassays in which neonates were allowed to orient within arenas to media with or without fungal tissue, and performance bioassays in which larvae were reared with and without A. flavus on potato dextrose agar (PDA) and a semidefined almond PDA diet to evaluate effects on development and pupal weight. Navel orangeworm larvae were attracted to A. flavus and developed faster in its presence, indicating a nutritional benefit to the caterpillars. Larvae reached pupation ∼33% faster on diet containing A. flavus, and pupal weights were ∼18% higher for males and ∼13% higher for females on this diet. Our findings indicate that A. flavus plays an important role in larval orientation and development on infected hosts. The preference-performance relationship between navel orangeworms and Aspergillus flavus is consistent with a facultative mutualism that has broad implications for pest management efforts and basic understanding of Lepidoptera-plant interactions.

Effect of Piperonyl Butoxide on the Toxicity of Four Classes of Insecticides to Navel Orangeworm (Amyelois transitella) (Lepidoptera: Pyralidae).

Amyelois transitella (Walker) (Lepidoptera: Pyralidae), the navel orangeworm, is a highly polyphagous economic pest of almond, pistachio, and walnut crops in California. Increasing demand for these crops and their rising economic value has resulted in substantial increases of insecticide applications to reduce damage to acceptable levels. The effects of piperonyl butoxide (PBO), a methylenedioxyphenyl compound that can act as a synergist by inhibiting cytochrome P450-mediated detoxification on insecticide metabolism by A. transitella, were examined in a series of feeding bioassays with first-instar A. transitella larvae from a laboratory strain. PBO, however, can have a variety of effects on metabolism, including inhibition of glutathione-S-transferases and esterases and induction of P450s. In our study, PBO synergized the toxicity of acetamiprid, λ-cyhalothrin, and spinosad, suggesting possible involvement of P450s in their detoxification. In contrast, PBO interacted antagonistically with the organophosphate insecticide chlorpyrifos, reducing its toxicity, an effect consistent with inhibition of P450-mediated bioactivation of this pesticide. The toxicity of the anthranilic diamide insecticide chlorantraniliprole was not altered by PBO, suggestive of little or no involvement of P450-mediated metabolism in its detoxification. Because a population of navel orangeworm in Kern County, CA, has already acquired resistance to the pyrethroid insecticide bifenthrin through enhanced P450 activity, determining the effect of adding a synergist such as PBO on detoxification of all insecticide classes registered for use in navel orangeworm management can help to develop rotation practices that may delay resistance acquisition or to implement alternative management practices where resistance is likely to evolve.

Mechanism of Resistance Acquisition and Potential Associated Fitness Costs in Amyelois transitella (Lepidoptera: Pyralidae) Exposed to Pyrethroid Insecticides.

The polyphagous navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive pest of nut crops, including almonds and pistachios, in California orchards. Management of this insect has typically been a combination of cultural controls and insecticide use, with the latter increasing substantially along with the value of these commodities. Possibly associated with increased insecticide use, resistance has been observed recently in navel orangeworm populations in Kern County, California. In studies characterizing a putatively pyrethroid-resistant strain (R347) of navel orangeworm, susceptibility to bifenthrin and ß-cyfluthrin was compared with that of an established colony of susceptible navel orangeworm. Administration of piperonyl butoxide and S,S,S-tributyl phosphorotrithioate in first-instar feeding bioassays with the pyrethroids bifenthrin and ß-cyfluthrin produced synergistic effects and demonstrated that cytochrome P450 monooxygenases and carboxylesterases contribute to resistance in this population. Resistance is therefore primarily metabolic and likely the result of overexpression of specific cytochrome P450 monooxygenases and carboxylesterase genes. Resistance was assessed by median lethal concentration (LC50) assays and maintained across nine generations in the laboratory. Life history trait comparisons between the resistant strain and susceptible strain revealed significantly lower pupal weights in resistant individuals reared on the same wheat bran-based artificial diet across six generations. Time to second instar was greater in the resistant strain than the susceptible strain, although overall development time was not significantly different between strains. Resistance was heritable and may have an associated fitness cost, which could influence the dispersal and expansion of resistant populations in nut-growing areas in California.

Aflatoxin B1: toxicity, bioactivation and detoxification in the polyphagous caterpillar Trichoplusia ni.

Trichoplusia ni caterpillars are polyphagous foliage-feeders and rarely likely to encounter aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB1, we evaluated the toxicity of AFB1 to T. ni caterpillars at different developmental stages and found that AFB1 tolerance significantly increases with larval development. Diet incorporation of AFB1 at 1 µg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3 µg/g AFB1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases (P450s), reduced the toxicity of AFB1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as flavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFB1 toxicity, suggesting that P450s are also involved in AFB1 detoxification in T. ni.

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella).

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b(5) on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.

Role of cysteine proteinase inhibitors in preference of Japanese beetles (Popillia japonica) for soybean (Glycine max) leaves of different ages and grown under elevated CO2.

Elevated levels of CO(2), equivalent to those projected to occur under global climate change scenarios, increase the susceptibility of soybean foliage to herbivores by down-regulating the expression of genes related to the defense hormones jasmonic acid and ethylene; these in turn decrease the gene expression and activity of cysteine proteinase inhibitors (CystPIs), the principal antiherbivore defenses in foliage. To examine the effects of elevated CO(2) on the preference of Japanese beetle (JB; Popillia japonica) for leaves of different ages within the plant, soybeans were grown at the SoyFACE facility at the University of Illinois at Urbana-Champaign. When given a choice, JB consistently inflicted greater levels of damage on older leaves than on younger leaves, and there was a trend for a greater preference for young leaves grown under elevated CO(2) compared to those grown under ambient CO(2). More heavily damaged older leaves and those grown under elevated CO(2) had reduced CystPI activity, and JB that consumed leaves with lower CystPI activity had correspondingly greater gut proteinase activity. Younger leaves with higher CystPI activity and photosynthetic rates may contribute disproportionately to plant fitness and are more protected against herbivore attack than older foliage. Cysteine proteinase inhibitors are potent defenses against JB, and the effectiveness of this defense is modulated by growth under elevated CO(2) as well as leaf position.

Ecological significance of induction of broad-substrate cytochrome P450s by natural and synthetic inducers in Helicoverpa zea.

The polyphagous corn earworm Helicoverpa zea relies on cytochrome P450 monooxygenases with broad substrate specificities to cope with the wide diversity of phytochemicals it encounters among its numerous host plants. These enzymes also contribute to the ability of this insect to tolerate toxins from sources other than its hosts, including microbial and synthetic toxins. Although upregulation of xenobiotic-metabolizing P450s in some herbivorous insects is closely linked to host plant toxins, transcriptional and/or post-transcriptional regulation of detoxification in this polyphagous species also appears to be relatively unspecialized. Reverse transcription polymerase chain reaction and metabolic analyses indicate that rare and infrequently encountered phytochemicals, as well as synthetic substances, can enhance metabolic activity in an adaptive fashion against both natural and synthetic toxins.

Aflatoxin B1 detoxification by CYP321A1 in Helicoverpa zea.

The polyphagous corn earworm Helicoverpa zea frequently encounters aflatoxins, mycotoxins produced by the pathogens Aspergillus flavus and A. parasiticus, which infect many of this herbivore's host plants. While aflatoxin B1 metabolism by midgut enzymes isolated from fifth instars feeding on control diets was not detected, this compound was metabolized by midgut enzymes isolated from larvae consuming diets supplemented with xanthotoxin, coumarin, or indole-3-carbinol, phytochemicals that are likely to co-occur with aflatoxin in infected host plants. Of the two metabolites generated, the main derivative identified in midguts induced with these chemicals and in reactions containing heterologously expressed CYP321A1 was aflatoxin P1 (AFP1), an O-demethylated product of AFB1. RT-PCR gel blots indicated that the magnitude of CYP321A1 transcript induction by these chemicals is associated with the magnitude of increase in the metabolic activities of induced midgut enzymes (coumarin>xanthotoxin>indole 3-carbinol). These results indicate that induction of P450s, such as CYP321A1, plays an important role in reducing AFB1 toxicity to H. zea. Docking of AFB1 in the molecular models of CYP321A1 and CYP6B8 highlights differences in their proximal catalytic site volumes that allow only CYP321A1 to generate the AFP1 metabolite.

Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects.

Elevated levels of atmospheric carbon dioxide (CO2), a consequence of anthropogenic global change, can profoundly affect the interactions between crop plants and insect pests and may promote yet another form of global change: the rapid establishment of invasive species. Elevated CO2 increased the susceptibility of soybean plants grown under field conditions to the invasive Japanese beetle (Popillia japonica) and to a variant of western corn rootworm (Diabrotica virgifera virgifera) resistant to crop rotation by down-regulating gene expression related to defense signaling [lipoxygenase 7 (lox7), lipoxygenase 8 (lox8), and 1-aminocyclopropane-1-carboxylate synthase (acc-s)]. The down-regulation of these genes, in turn, reduced the production of cysteine proteinase inhibitors (CystPIs), which are specific deterrents to coleopteran herbivores. Beetle herbivory increased CystPI activity to a greater degree in plants grown under ambient than under elevated CO2. Gut cysteine proteinase activity was higher in beetles consuming foliage of soybeans grown under elevated CO2 than in beetles consuming soybeans grown in ambient CO2, consistent with enhanced growth and development of these beetles on plants grown in elevated CO2. These findings suggest that predicted increases in soybean productivity under projected elevated CO2 levels may be reduced by increased susceptibility to invasive crop pests.

Transcriptional profiling reveals elevated CO2 and elevated O3 alter resistance of soybean (Glycine max) to Japanese beetles (Popillia japonica).

The accumulation of CO2 and O3 in the troposphere alters phytochemistry which in turn influences the interactions between plants and insects. Using microarray analysis of field-grown soybean (Glycine max), we found that the number of transcripts in the leaves affected by herbivory by Japanese beetles (Popillia japonica) was greater when plants were grown under elevated CO2, elevated O3 and the combination of elevated CO2 plus elevated O3 than when grown in ambient atmosphere. The effect of herbivory on transcription diminished strongly with time (<1% of genes were affected by herbivory after 3 weeks), and elevated CO2 interacted more strongly with herbivory than elevated O3. The majority of transcripts affected by elevated O3 were related to antioxidant metabolism. Constitutive levels and the induction by herbivory of key transcripts associated with defence and hormone signalling were down-regulated under elevated CO2; 1-aminocyclopropane-1-carboxylate (ACC) synthase, lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), chalcone synthase (CHS), polyphenol oxidase (PPO) and cysteine protease inhibitor (CystPI) were lower in abundance compared with levels under ambient conditions. By suppressing the ability to mount an effective defence, elevated CO2 may decrease resistance of soybean to herbivory.

Foliage of oaks grown under elevated CO2 reduces performance of Antheraea polyphemus (Lepidoptera: Saturniidae).

To understand how the increase in atmospheric CO2 from human activity may affect leaf damage by forest insects, we examined host plant preference and larval performance of a generalist herbivore, Antheraea polyphemus Cram., that consumed foliage developed under ambient or elevated CO2. Larvae were fed leaves from Quercus alba L. and Quercus velutina Lam. grown under ambient or plus 200 microl/liter CO2 using free air carbon dioxide enrichment (FACE). Lower digestibility of foliage, greater protein precipitation capacity in frass, and lower nitrogen concentration of larvae indicate that growth under elevated CO2 reduced the food quality of oak leaves for caterpillars. Consuming leaves of either oak species grown under elevated CO2 slowed the rate of development of A. polyphemus larvae. When given a choice, A. polyphemus larvae preferred Q. velutina leaves grown under ambient CO2; feeding on foliage of this species grown under elevated CO2 led to reduced consumption, slower growth, and greater mortality. Larvae compensated for the lower digestibility of Q. alba leaves grown under elevated CO2 by increasing the efficiency of conversion of ingested food into larval mass. Despite equivalent consumption rates, larvae grew larger when they consumed Q. alba leaves grown under elevated compared with ambient CO2. Reduced consumption, slower growth rates, and increased mortality of insect larvae may explain lower total leaf damage observed previously in plots in this forest exposed to elevated CO2. By subtly altering aspects of leaf chemistry, the ever-increasing concentration of CO2 in the atmosphere will change the trophic dynamics in forest ecosystems.

Allelochemical induction of cytochrome P450 monooxygenases and amelioration of xenobiotic toxicity in Helicoverpa zea.

Polyphagous herbivores encounter allelochemicals as complex mixtures in their host plants, and the toxicity of an individual compound may be influenced by the chemical matrix in which it is encountered. Certain plant constituents may reduce toxicity of cooccurring compounds by inducing detoxification systems, including cytochrome P450s, which can metabolize a broad range of substances. The polyphagous corn earworm Helicoverpa zea encounters a diversity of plant allelochemicals in its many host plants and, as well, can encounter aflatoxins, mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus that infect damaged grains. Dietary supplementation of each of three plant allelochemicals that are frequently (coumarin, COU), occasionally (indole-3-carbinol, 13C), or rarely (xanthotoxin, XAN) encountered by H. zea larvae substantially reduced the toxicity of aflatoxin B1 (AFB1) to H. zea. Compared to fourth instars on diets containing 1 microg/g AFB1 that failed to develop and pupate, fourth instars on diets containing I3C and XAN increased in mass by 216.1 and 700% after 6 days, and pupated at rates of 40 and 88%, respectively. Diets containing COU or XAN also significantly reduced the mortality rates of caterpillars exposed to the insecticides, diazinon and carbaryl. Diets containing COU and XAN increased CYP6B8 transcripts 2.6-fold; CYP321A1 transcripts increased 20.7, 8.3, and 10.6-fold in response to COU, I3C, and XAN, respectively. These results indicate that consumption of plant allelochemicals can ameliorate toxicity of natural and synthetic toxins encountered by insects, and they suggest that P450s induced by these allelochemicals contribute to detoxification of these chemicals in H. zea.

Toxicity of aflatoxin B1 to Helicoverpa zea and bioactivation by cytochrome P450 monooxygenases.

Infestation of corn (Zea mays) by corn earworm (Helicoverpa zea) predisposes the plant to infection by Aspergillus fungi and concomitant contamination with the carcinogenic mycotoxin aflatoxin B1 (AFB1). Although effects of ingesting AFB1 are well documented in livestock and humans, the effects on insects that naturally encounter this mycotoxin are not as well defined. Toxicity of AFB1 to different stages of H. zea (first, third, and fifth instars) was evaluated with artificial diets containing varying concentrations. Although not acutely toxic at low concentrations (1-20 ng/g), AFB1 had significant chronic effects, including protracted development, increased mortality, decreased pupation rate, and reduced pupal weight. Sensitivity varied with developmental stage; whereas intermediate concentrations (200 ng/g) caused complete mortality in first instars, this same concentration had no detectable adverse effects on larvae encountering AFB1 in fifth instar. Fifth instars consuming AFB1 at higher concentrations (1 microg/g), however, displayed morphological deformities at pupation. That cytochrome P450 monooxygenases (P450s) are involved in the bioactivation of aflatoxin in this species is evidenced by the effects of piperonyl butoxide (PBO), a known P450 inhibitor, on toxicity; whereas no fourth instars pupated in the presence of 1 mug/g AFB1 in the diet, the presence of 0.1% PBO increased the pupation rate to 71.7%. Pupation rates of both fourth and fifth instars on diets containing 1 mug/g AFB1 also increased significantly in the presence of PBO. Effects of phenobarbital, a P450 inducer, on AFB1 toxicity were less dramatic than those of PBO. Collectively, these findings indicate that, as in many other vertebrates and invertebrates, toxicity of AFB1 to H. zea results from P450-mediated metabolic bioactivation.

Cytochrome P450-mediated metabolism of xanthotoxin by Papilio multicaudatus.

Within the genus Papilio, the P. glaucus group contains the most polyphagous Papilio species within the Papilionidae. The majority of Papilio species are associated with hostplants in the families Rutaceae and Apiaceae, and characterizing most are secondary metabolites called furanocoumarins. Recent phylogenetic studies suggest that furanocoumarin metabolism is an ancestral trait, with the glaucus group derived from ancestors associated with furanocoumarin-containing Rutaceae. In this study, we examined this relationship by conducting a gravimetric analysis of growth that used various concentrations of the furanocoumarin xanthotoxin. Papilio multicaudatus, the putative ancestor of the glaucus group, includes at least one furanocoumarin-containing rutaceous species among its hostplants; this species can consume leaf tissue containing up to 0.3% xanthotoxin with no detectable effect on relative growth rate, relative consumption rate, or efficiency of conversion of ingested food. As is the case for other Papilio species, xanthotoxin metabolism is mediated by cytochrome P450 monooxygenases (P450s). Ingestion of xanthotoxin by ultimate instar P. multicaudatus increases activity up to 30-fold in a dose-dependent fashion. Midguts of induced larvae can also effectively metabolize six other furanocoumarins, including both linear (bergapten, isopimpinellin, imperatorin) and angular (angelicin, sphondin) forms. A metabolite of xanthotoxin in the frass from xanthotoxin-treated larvae, identified as 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid by MS-MS and NMR analyses, is identical to one from the frass of P. polyxenes. The occurrence of this metabolite in two swallowtails and the presence of a second metabolite of xanthotoxin, 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol in the frass of both P. polyxenes and Depressaria pastinacella are consistent with the suggestion that lepidopterans share as the first step of xanthotoxin metabolism the P450-mediated epoxidation of the furan ring 2'-3' double bond.

Structural and functional divergence of insect CYP6B proteins: From specialist to generalist cytochrome P450.

How polyphagous herbivores cope with the diversity and unpredictability of plant defenses remains largely unknown at both the genetic and molecular levels. To examine whether generalist counterdefense enzymes are structurally more flexible and functionally more diverse, two counterdefensive allelochemical-metabolizing cytochrome P450 proteins, CYP6B1 from the specialist Papilio polyxenes, feeding on furanocoumarin-containing plants, and CYP6B8 from the generalist Helicoverpa zea, feeding on hundreds of host plant species, are compared structurally and functionally. Molecular modeling indicates that CYP6B8 has more flexible overall folding, a more elastic catalytic pocket, and one more substrate access channel than CYP6B1. Baculovirus-mediated expression of the CYP6B8 and CYP6B1 proteins demonstrates that CYP6B8 metabolizes six biosynthetically diverse plant allelochemicals (xanthotoxin, quercetin, flavone, chlorogenic acid, indole-3-carbinol, and rutin) and three insecticides (diazinon, cypermethrin, and aldrin), whereas CYP6B1 metabolizes only two allelochemicals (xanthotoxin and flavone) and one insecticide (diazinon) of those tested. These results indicate that generalist counterdefense proteins are capable of accepting a more structurally diverse array of compounds compared with specialist counterdefense proteins.