More than 10 years after commercialization, Vip3A-expressing MIR162 remains highly efficacious in controlling major Lepidopteran maize pests: laboratory resistance selection versus field reality.

MIR162, a maize event that expresses Vip3Aa20 (Vip3A) approved for commercial cultivation around 2010, has been excellent for control of major Lepidopteran pests. However, development of fall armyworm (FAW) resistance to Vip3A is a serious concern. Resistant colonies selected in the laboratory can serve as valuable tools not only for better understanding of Vip3A's mode of action (MOA) and mechanism of resistance (MOR) but also for screening novel leads of new MOA that will help control FAW in case resistance to Vip3A in the field becomes a reality. We selected a Vip3A-resistant FAW strain, FAWVip3AR, by subjecting a FAW founder population containing field genetics to Vip3A exposure. FAWVip3AR had >9800-fold resistance to Vip3A by diet surface overlay bioassays and resistance was stable. Feeding bioassays using detached leaf tissues or whole plants indicated that FAWVip3AR larvae readily fed and completed the full life cycle on Vip3A-expressing MIR162 maize plants and leaf tissues that killed 100% of susceptible larvae. Yet, FAWVip3AR faced at least two challenges. First, FAWVip3AR suffered an apparent disadvantage (incomplete resistance) when feeding on MIR162 in comparison to FAWVip3AR feeding on Vip3A-free isoline AX5707 maize; and second, FAWVip3AR showed a fitness costs in comparison to a Vip3A-susceptible strain when both fed on AX5707. We also demonstrated that, >10 years after commercialization, MIR162 and Vip3A remain highly efficacious against field populations of three major Lepidopteran pests from different geographic locations and FAW strains resistant to other Bacillus thuringiensis (Bt) toxins that are currently on the market.

eCry1Gb.1Ig, A Novel Chimeric Cry Protein with High Efficacy against Multiple Fall Armyworm (Spodoptera frugiperda) Strains Resistant to Different GM Traits.

Spodoptera frugiperda (fall armyworm, FAW) is one of the most devastating insect pests to corn and soybean production in the Americas and is rapidly expanding its range worldwide. It is known to be hard to control either by chemical insecticide applications or by GM. Although the use of GM traits can be an effective way to control this pest, it is very rare to find native insecticidal proteins that provide the necessary level of FAW control in crop fields where FAW pressure and damage are high. Insecticidal Cry proteins sourced from Bacillus thuringiensis have been heavily utilized in the development of crops with GM traits; however, it is increasingly difficult to identify Cry proteins with unique modes of action. Protein engineering via a phylogenetically guided Cry protein domain swapping approach enabled us to discover novel chimeric Cry proteins engineered from inactive parent sequences. Some of these chimeras show excellent efficacy against key biotypes of FAW from Brazil and North America. In this study, we characterized a Cry-based chimera eCry1Gb.1Ig that is a very potent FAW toxin. eCry1Gb.1Ig showed high efficacy against multiple FAW strains that are resistant to various traits, including Cry1Fa, Vip3Aa and Cry1A.105/Cry2Ab. These results clearly indicate that the FAW strains resistant to Cry1Fa, Vip3Aa or Cry1A.105/Cry2Ab demonstrate no cross-resistance to eCry1Gb.1Ig and strongly suggest that eCry1Gb.1Ig acts through a novel mode of action compared to the existing traits. In addition to its FAW activity, eCry1Gb.1Ig has also been shown to control Chrysodeixis includens (soybean looper, SBL) and Anticarsia gemmatalis (velvetbean caterpillar, VBC), which are significant pests of soybean. When eCry1Gb.1Ig was introduced into corn and soybean crops, transgenic events showed strong efficacy against FAW, SBL and VBC, but no adverse plant phenotypes. This suggests that the in planta expression of the eCry1Gb.1Ig protein does not compromise plant growth or reproduction and can protect plants from FAW-related damage. Therefore, this valuable discovery will provide a differentiating FAW control trait that will give growers another tool to help them reduce yield loss due to FAW.

Resistance Allele Frequency to Cry1Ab and Vip3Aa20 in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Louisiana and Three Other Southeastern U.S. States.

The corn earworm/bollworm, Helicoverpa zea (Boddie), is a pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1Ab and Vip3Aa20 are two common Bt toxins that are expressed in transgenic maize. The objective of this study was to determine the resistance allele frequency (RAF) to Cry1Ab and Vip3Aa20 in H. zea populations that were collected during 2018 and 2019 from four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. By using a group-mating approach, 104 F2 iso-lines of H. zea were established from field collections with most iso-lines (85) from Louisiana. These F2 iso-lines were screened for resistance alleles to Cry1Ab and Vip3Aa20, respectively. There was no correlation in larval survivorship between Cry1Ab and Vip3Aa20 when the iso-lines were exposed to these two toxins. RAF to Cry1Ab maize was high (0.256) and the RAFs were similar between Louisiana and the other three states and between the two sampling years. In contrast, no functional major resistance allele (RA) that allowed resistant insects to survive on Vip3Aa20 maize was detected and the expected RAF of major RAs with 95% probability was estimated to 0 to 0.0073. However, functional minor RAs to Vip3Aa20 maize were not uncommon; the estimated RAF for minor alleles was 0.028. The results provide further evidence that field resistance to Cry1Ab maize in H. zea has widely occurred, while major RAs to Vip3Aa20 maize are uncommon in the southeastern U.S. region. Information that was generated from this study should be useful in resistance monitoring and refinement of resistance management strategies to preserve Vip3A susceptibility in H. zea.

Populations of Helicoverpa zea (Boddie) in the Southeastern United States are Commonly Resistant to Cry1Ab, but Still Susceptible to Vip3Aa20 Expressed in MIR 162 Corn.

The corn earworm, Helicoverpa zea (Boddie), is a major pest targeted by pyramided Bacillus thuringiensis (Bt) corn and cotton in the U.S. Cry1Ab is one of the first insecticidal toxins used in Bt crops, while Vip3A is a relatively new toxin that has recently been incorporated into Cry corn with event MIR 162 and Cry cotton varieties to generate pyramided Bt traits targeting lepidopteran pests including H. zea. The objectives of this study were to determine the current status and distribution of the Cry1Ab resistance, and evaluate the susceptibility to Vip3Aa20 expressed in MIR 162 corn in H. zea in the southeastern U.S. During 2018 and 2019, 32 H. zea populations were collected from non-Bt corn (19 populations), Cry corn (12), and Cry/Vip3A cotton (1) across major corn areas in seven southeastern states of the U.S. Susceptibility of these populations to Cry1Ab and Vip3Aa20 was determined using diet-overlay bioassays. Compared to a known susceptible insect strain, 80% of the field populations were 13- to >150-fold resistant to Cry1Ab, while their response to Vip3Aa20 ranged from >11-fold more susceptible to 9-fold more tolerant. Mean susceptibility to each Bt toxin was not significantly different between the two groups of the populations collected from non-Bt and Bt crops, as well as between the two groups of the populations collected during 2018 and 2019. The results show that resistance to Cry1Ab in H. zea is widely distributed across the region. However, the Cry1Ab-resistant populations are not cross-resistant to Vip3Aa20, and H. zea in the region is still susceptible to the Vip3Aa20 toxin. Vip3Aa20 concentrations between 5 and 10 µg/cm2 may be used as diagnostic concentrations for susceptibility monitoring in future. Additional studies are necessary to elucidate the impact of the selection with Bt corn on resistance evolution in H. zea to Vip3A cotton in the U.S.

A Simple and Sensitive Plant-Based Western Corn Rootworm Bioassay Method for Resistance Determination and Event Selection.

We report here a simple and sensitive plant-based western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), bioassay method that allows for examination of multiple parameters for both plants and insects in a single experimental setup within a short duration. For plants, injury to roots can be visually examined, fresh root weight can be measured, and expression of trait protein in plant roots can be analyzed. For insects, in addition to survival, larval growth and development can be evaluated in several aspects including body weight gain, body length, and head capsule width. We demonstrated using the method that eCry3.1Ab-expressing 5307 corn was very effective against western corn rootworm by eliciting high mortality and significantly inhibiting larval growth and development. We also validated that the method allowed determination of resistance in an eCry3.1Ab-resistant western corn rootworm strain. While data presented in this paper demonstrate the usefulness of the method for selection of events of protein traits and for determination of resistance in laboratory populations, we envision that the method can be applied in much broader applications.

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.

Two insulin-like peptide family members from the mosquito Aedes aegypti exhibit differential biological and receptor binding activities.

Insects encode multiple ILPs but only one homolog of the vertebrate IR that activates the insulin-signaling pathway. However, it remains unclear whether all insect ILPs are high affinity ligands for the IR or have similar biological functions. The yellow fever mosquito, Aedes aegypti, encodes eight ILPs with prior studies strongly implicating ILPs from the brain in regulating metabolism and the maturation of eggs following blood feeding. Here we addressed whether two ILP family members expressed in the brain, ILP4 and ILP3, have overlapping functional and receptor binding activities. Our results indicated that ILP3 exhibits strong insulin-like activity by elevating carbohydrate and lipid storage in sugar-fed adult females, whereas ILP4 does not. In contrast, both ILPs exhibited dose-dependent gonadotropic activity in blood-fed females as measured by the stimulation of ovaries to produce ecdysteroids and the uptake of yolk by primary oocytes. Binding studies using ovary membranes indicated that ILP4 and ILP3 do not cross compete; a finding further corroborated by cross-linking and immunoblotting experiments showing that ILP3 binds the MIR while ILP4 binds an unknown 55kDa membrane protein. In contrast, each ILP activated the insulin-signaling pathway in ovaries as measured by enhanced phosphorylation of Akt. RNAi and inhibitor studies further indicated that the gonadotropic activity of ILP4 and ILP3 requires the MIR and a functional insulin-signaling pathway. Taken together, our results indicate that two members of the Ae. aegypti ILP family exhibit partially overlapping biological activity and different binding interactions with the MIR.

Enhanced toxicity and induction of cytochrome P450s suggest a cost of "eavesdropping" in a multitrophic interaction.

The inducibility of cytochrome P450 monooxygenases (P450s) and other xenobiotic-metabolizing enzymes is thought to reflect material and energy costs of biosynthesis. Efforts to detect such costs of detoxification enzyme induction, however, have had mixed success. Although they are rarely considered, ecological costs of induction may be a more significant evolutionary constraint on herbivores than material and energy costs. Because some P450-mediated metabolic transformations are bioactivation reactions that increase, rather than reduce, toxicity, maintaining high levels of P450 activity places an organism at risk of greater mortality in the presence of compounds that are bioactivated. We show that P450 inducibility in the generalist moth Helicoverpa zea in response to plant signaling substances, an adaptive response in a ditrophic interaction between herbivore and plant, becomes detrimental in the presence of a third trophic association with a plant pathogen that produces aflatoxin, a toxin that can be bioactivated by P450s. Consumption of plant signaling molecules, such as methyl jasmonate (MeJA) and salicylic acid (SA) enhanced the toxicity of aflatoxin B1 (AFB1) to H. zea that resulted in substantially more damage to larval growth and development. Among the P450 transcripts already cloned from this organism, two in the CYP6B and CYP321A subfamilies are shown to be induced in response to MeJA and SA, suggesting that they may mediate some of the observed bioactivations.

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.

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT.

One of the challenges faced in malarial control is the acquisition of insecticide resistance that has developed in mosquitoes that are vectors for this disease. Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa, has over the years developed resistance to insecticides including dieldrin, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pyrethroids. Previous microarray studies using fragments of 230 An. gambiae genes identified five P450 loci, including CYP4C27, CYP4H15, CYP6Z1, CYP6Z2, and CYP12F1, that showed significantly higher expression in the DDT-resistant ZAN/U strain compared with the DDT-susceptible Kisumu strain. To predict whether either of the CYP6Z1 and CYP6Z2 proteins might potentially metabolize DDT, we generated and compared molecular models of these two proteins with and without DDT docked in their catalytic sites. This comparison indicated that, although these two CYP6Z proteins share high sequence identity, their metabolic profiles were likely to differ dramatically from the larger catalytic site of CYP6Z1, potentially involved in DDT metabolism, and the more constrained catalytic site of CYP6Z2, not likely to metabolize DDT. Heterologous expressions of these proteins have corroborated these predictions: only CYP6Z1 is capable of metabolizing DDT. Overlays of these models indicate that slight differences in the backbone of SRS1 and variations of side chains in SRS2 and SRS4 account for the significant differences in their catalytic site volumes and DDT-metabolic capacities. These data identify CYP6Z1 as one important target for inhibitor design aimed at inactivating insecticide-metabolizing P450s in natural populations of this malarial mosquito.

Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides.

Under continual exposure to naturally occurring plant toxins and synthetic insecticides, insects have evolved cytochrome P450 monooxygenases (P450s) capable of metabolizing a wide range of structurally different compounds. Two such P450s, CYP6B8 and CYP321A1, expressed in Helicoverpa zea (a lepidopteran) in response to plant allelochemicals and plant signaling molecules metabolize these compounds with varying efficiencies. While sequence alignments of these proteins indicate highly divergent substrate recognition sites (SRSs), homology models developed for them indicate that the two active site cavities have essentially the same volume with distinct shapes dictated by side-chain differences in SRS1 and SRS5. CYP6B8 has a narrower active site cavity extending from substrate access channel pw2a with a very narrow access to the ferryl oxygen atom. This predicted shape suggests that bulkier molecules bind further from the ferryl oxygen at positions that are not as effectively metabolized. In contrast, CYP321A1 is predicted to have a more spacious cavity allowing larger molecules to access the heme-bound oxygen. The metabolic profiles for several plant toxins (xanthotoxin, angelicin) and insecticides (cypermethrin, aldrin and diazinon) correlate well with these predictive models. The absence of Thr in the I helix of CYP321A1 and hydroxyl groups on many of its substrates suggests that this insect P450 mediates oxygen activation by a mechanism different from that employed by CYP107A1 and CYP158A1, which are two bacterial P450s also lacking Thr in their I helix, and most other P450s that contain Thr in their I helix.

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.

CYP6B1 and CYP6B3 of the black swallowtail (Papilio polyxenes): adaptive evolution through subfunctionalization.

Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6B1 and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases.

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.

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.

Inhibition of CYP6B1-mediated detoxification of xanthotoxin by plant allelochemicals in the black swallowtail (Papilio polyxenes).

The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular furanocoumarins are generally less phototoxic to many organisms than linear furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel.

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea.

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism.

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.