Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays.

As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.

Natural variation in maize aphid resistance is associated with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity.

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar-induced aphid resistance in maize.

Plants in nature have inducible defences that sometimes lead to targeted resistance against particular herbivores, but susceptibility to others. The metabolic diversity and genetic resources available for maize (Zea mays) make this a suitable system for a mechanistic study of within-species variation in such plant-mediated interactions between herbivores. Beet armyworms (Spodoptera exigua) and corn leaf aphids (Rhopalosiphum maidis) are two naturally occurring maize herbivores with different feeding habits. Whereas chewing herbivore-induced methylation of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) to form 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) promotes caterpillar resistance, lower DIMBOA-Glc levels favour aphid reproduction. Thus, caterpillar-induced DIMBOA-Glc methyltransferase activity in maize is predicted to promote aphid growth. To test this hypothesis, the impact of S. exigua feeding on R. maidis progeny production was assessed using seventeen genetically diverse maize inbred lines. Whereas aphid progeny production was increased by prior caterpillar feeding on lines B73, Ki11, Ki3 and Tx303, it decreased on lines Ky21, CML103, Mo18W and W22. Genetic mapping of this trait in a population of B73 × Ky21 recombinant inbred lines identified significant quantitative trait loci on maize chromosomes 1, 7 and 10. There is a transgressive segregation for aphid resistance, with the Ky21 alleles on chromosomes 1 and 7 and the B73 allele on chromosome 10 increasing aphid progeny production. The chromosome 1 QTL coincides with a cluster of three maize genes encoding benzoxazinoid O-methyltransferases that convert DIMBOA-Glc to HDMBOA-Glc. Gene expression studies and benzoxazinoid measurements indicate that S. exigua -induced responses in this pathway differentially affect R. maidis resistance in B73 and Ky21.

Resistance evolution to the first generation of genetically modified Diabrotica-active Bt-maize events by western corn rootworm: management and monitoring considerations.

Western corn rootworm (Diabrotica virgifera virgifera; WCR) is a major coleopteran maize pest in North America and the EU, and has traditionally been managed through crop rotation and broad-spectrum soil insecticides. Genetically modified Bt-maize offers an additional management tool for WCR and has been valuable in reducing insecticide use and increasing farm income. A concern is that the widespread, repeated, and exclusive deployment of the same Bt-maize transformation event will result in the rapid evolution of resistance in WCR. This publication explores the potential of WCR to evolve resistance to plant-produced Bt-toxins from the first generation of Diabrotica-active Bt-maize events (MON 863 and MON 88017, DAS-59122-7 and MIR604), and whether currently implemented risk management strategies to delay and monitor resistance evolution are appropriate. In twelve of the twelve artificial selection experiments reported, resistant WCR populations were yielded rapidly. Field-selected resistance of WCR to Cry3Bb1 is documented in some US maize growing areas, where an increasing number of cases of unexpected damage of WCR larvae to Bt-maize MON 88017 has been reported. Currently implemented insect resistance management measures for Bt-crops usually rely on the high dose/refuge (HDR) strategy. Evidence (including laboratory, greenhouse and field data) indicates that several conditions contributing to the success of the HDR strategy may not be met for the first generation of Bt-maize events and WCR: (1) the Bt-toxins are expressed heterogeneously at a low-to-moderate dose in roots; (2) resistance alleles may be present at a higher frequency than initially assumed; (3) WCR may mate in a non-random manner; (4) resistance traits could have non-recessive inheritance; and (5) fitness costs may not necessarily be associated with resistance evolution. However, caution must be exercised when extrapolating laboratory and greenhouse results to field conditions. Model predictions suggest that a 20 % refuge of non-Diabrotica-active Bt-maize can delay resistance evolution in WCR under certain conditions. This publication concludes that further research is needed to resolve the remaining scientific uncertainty related to the appropriateness of the HDR in delaying resistance evolution in WCR, resistance monitoring is essential to detect early warning signs indicating resistance evolution in the field, and that integrated pest management reliant on multiple tactics should be deployed to ensure effective long-term corn rootworm management and sustainable use of Bt-maize.

Increased survival of western corn rootworm on transgenic corn within three generations of on-plant greenhouse selection.

To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby "refuges" of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this "high-dose" criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1-4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC(50) was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management.

Selection for resistance to mCry3A-expressing transgenic corn in western corn rootworm.

To investigate the development of resistance to mCry3A, a laboratory colony of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was established from field survivors of mCry3A-expressing (MIR604) corn, Zea mays L. Feral adults emerging from MIR604 (selected) and isoline (control) field plots were collected and returned to the laboratory. Progeny of each colony was reared one generation on isoline corn and then crossed reciprocally with a nondiapausing colony. The resulting nondiapausing progeny were then reared on greenhouse corn in accordance with the wild type parent's origin (on MIR604 or isoline corn). After four, seven, and 10 total generations of selection, the resistance ratio of the selected colony was 0.5, 4.3, and 15.4 in terms of lethal concentration (LC)50 values in toxicity assays, with the latter two LC50 values being significant. After seven generations of selection in total, selected and control colonies were screened on MIR604 and isoline corn under field conditions. There was a significant colony x corn pedigree interaction in terms of plant damage. There was no significant difference in damage between MIR604 and isoline corn, whereas this difference was significant for the control colony. After 14 generations of selection, a seedling bioassay was performed. Again, there was a significant colony x corn pedigree interaction, this time in terms of the number of larvae recovered. There was no significant difference in the number of larvae recovered from MIR604 and isoline corn for the selected colony, whereas this difference was significant for the control colony, although larval size was greater on isoline corn for both colonies. Resistance has developed in western corn rootworm laboratory colonies to all Bt proteins currently registered for corn rootworm management, which emphasizes the importance of adhering to resistance management plans for maintaining product efficacy.

Development of a nondiapausing strain of northern corn rootworm with rearing techniques for both diapausing and nondiapausing strains.

The northern corn rootworm, Diabrotica barberi Smith & Lawrence, has a univoltine life cycle that typically produces one generation a year. When rearing the northern corn rootworm in the laboratory, in order to break diapause, it is necessary to expose eggs to a five month cold period before raising the temperature. By selective breeding of the small fraction of eggs that hatched without cold within 19-32 days post oviposition, we were able to develop a non-diapausing colony of the northern corn rootworm within five generations of selection. Through selection, the percentages of adult emergence from egg hatch without exposure to cold treatment significantly increased from 0.52% ± 0.07 at generation zero to 29.0% ± 2.47 at generation eight. During this process, we developed an improved method for laboratory rearing of both the newly developed non-diapausing strain as well as the diapausing strain. The development of the non-diapausing colony along with the improvements to the rearing system will allow researchers to produce up to six generations of the northern corn rootworm per year, which would facilitate research and advance our knowledge of this pest at an accelerated rate.

Density-dependent and density-independent mortality of the western corn rootworm: impact on dose calculations of rootworm-resistant Bt corn.

The percentage of viable eggs of the western corn rootworm, Diabrotica virgifera virgifera LeConte, which survived to the adult stage was evaluated for the effect of egg density in 2005 and 2007 in central Missouri. In 2005, each plot was 2.44 by 3.05 m and contained 64 maize (corn), Zea mays L., plants. In 2007, plots were 3.05 by 3.05 m and again contained 64 corn plants. Seven egg densities (2,400, 1,200, 600, 300, 100, 50, and 25 viable eggs per 30.5 cm) were evaluated with four to six replications in each year in a completely randomized design. In 2007 only, an additional row was infested near each plot to evaluate plant damage. In both years, there was no correlation of infestation level and percentage of emergence between infestation levels of 25-600 viable eggs per 30.5 cm, indicating that density-dependent mortality did not occur at these egg densities. In 2005, 8.04% of the viable eggs established on a corn plant and produced an adult at these lower infestation rates. In 2007, this value was 2.9%. Regardless of egg density, approximately 92-97% failed to establish and produce adults (density-independent mortality). In 2005 and in the combined analysis, as viable egg densities increased from 600 to 2400 per 30.5 cm there was a significant decrease in percentage of emergence. In a broken line analysis of the 2005 data, the point where density-dependent mortality began in the combined analysis was 851 eggs per 30.5 cm with a 95% confidence interval from 678 to 1024. That year density-dependent mortality was important at high infestations and killed 54.4% of those larvae that successfully established on a plant at the highest egg density. However, little or no density-dependent mortality occurred at infestation levels <850 viable eggs per 30.5 cm in either year of the study. Combining data from both years with all previously published data in a broken line analysis indicated that density-dependent mortality began at approximately 800 viable eggs per 30.5 cm. These data are discussed in terms of dose calculations for products targeting the western corn rootworm.

Mortality of western corn rootworm larvae on MIR604 transgenic maize roots: field survivorship has no significant impact on survivorship of F1 progeny on MIR604.

Mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to MIR604 transgenic corn, Zea mays L., expressing the modified Cry3A (mCry3A) protein relative to survivorship on corn with the same genetic background without the gene (isoline corn) was evaluated at three Missouri sites in both 2005 and 2006. We made these comparisons by using wild-type western corn rootworm at three different egg densities (6,000, 3,000, and 1,500 eggs per m) so that the role of density-dependent mortality would be known. The mortality due to the mCry3A protein was 94.88% when averaged across all environments and both years. Fifty percent emergence of beetles was delayed approximately 5.5 d. Beetles were kept alive and their progeny evaluated on MIR604 and isoline corn in the greenhouse to determine whether survivorship on MIR604 in the field for one generation increased survivorship on MIR604 in the greenhouse in the subsequent generation. There was no significant difference in survivorship on MIR604 in greenhouse assays between larvae whose parents survived isoline and larvae whose parents survived MIR604 in the field the previous generation, indicating that many susceptible beetles survived MIR604 in the field the previous season along with any potentially resistant beetles. The data are discussed in terms of rootworm insect resistance management.

Population growth of soybean aphid, Aphis glycines, under varying levels of predator exclusion.

Although soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), has caused economic damage in several Midwestern states, growers in Missouri have experienced relatively minor damage. To evaluate whether existing predatory insect populations are capable of suppressing or preventing soybean aphid population growth or establishment in Missouri, a predator exclusion study was conducted to gauge the efficacy of predator populations. Three levels of predator exclusion were used; one that excluded all insects (small mesh), one that excluded insects larger than thrips (medium mesh), and one that excluded insects larger than Orius insidiosus (Say) (Hemiptera: Anthocoridae), a principal predator (large mesh). Along with manipulating predator exposure, timing of aphid arrival (infestation) was manipulated. Three infestation times were studied; vegetative (V5), beginning bloom (R1), and beginning pod set (R3). Timing of aphid and predator arrival in a soybean field may affect the soybean aphid's ability to establish and begin reproducing. Cages infested at V5 and with complete predator exclusion reached economic threshold within two weeks, while cages with predators reached economic threshold in four and a half weeks. Cages infested at R1 with complete predator exclusion reached economic threshold within five weeks; cages with predators reached economic threshold within six weeks. Cages infested at R3 never reached threshold (with or without predators). The predator population in Missouri seems robust, capable of depressing the growth of soybean aphid populations once established, and even preventing establishment when the aphid arrived late in the field.

Differential gene expression in response to eCry3.1Ab ingestion in an unselected and eCry3.1Ab-selected western corn rootworm (Diabrotica virgifera virgifera LeConte) population.

Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR) is one of the most destructive pests in the U.S. Corn Belt. Transgenic maize lines expressing various Cry toxins from Bacillus thuringiensis have been adopted as a management strategy. However, resistance to many Bt toxins has occurred. To investigate the mechanisms of Bt resistance we carried out RNA-seq using Illumina sequencing technology on resistant, eCry3.1Ab-selected and susceptible, unselected, whole WCR neonates which fed on seedling maize with and without eCry3.1Ab for 12 and 24 hours. In a parallel experiment RNA-seq experiments were conducted when only the midgut of neonate WCR was evaluated from the same treatments. After de novo transcriptome assembly we identified differentially expressed genes (DEGs). Results from the assemblies and annotation indicate that WCR neonates from the eCry3.1Ab-selected resistant colony expressed a small number of up and down-regulated genes following Bt intoxication. In contrast, unselected susceptible WCR neonates expressed a large number of up and down-regulated transcripts in response to intoxication. Annotation and pathway analysis of DEGs between susceptible and resistant whole WCR and their midgut tissue revealed genes associated with cell membrane, immune response, detoxification, and potential Bt receptors which are likely related to eCry3.1Ab resistance. This research provides a framework to study the toxicology of Bt toxins and mechanism of resistance in WCR, an economically important coleopteran pest species.

Survey of bacteria associated with western corn rootworm life stages reveals no difference between insects reared in different soils.

Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a serious pest of maize (Zea mays L.) in North America and parts of Europe. With most of its life cycle spent in the soil feeding on maize root tissues, this insect is likely to encounter and interact with a wide range of soil and rhizosphere microbes. Our knowledge of the role of microbes in pest management and plant health remains woefully incomplete, yet that knowledge could play an important role in effective pest management strategies. For this study, insects were reared on maize in soils from different locations. Insects from two different laboratory colonies (a diapausing and a non-diapausing colony) were sampled at each life stage to determine the possible core bacteriome. Additionally, soil was sampled at each life stage and resulting bacteria were identified to determine the possible contribution of soil to the rootworm bacteriome, if any. We analyzed the V4 hypervariable region of bacterial 16S rRNA genes with Illumina MiSeq to survey the different species of bacteria associated with the insects and the soils. The bacterial community associated with insects was significantly different from that in the soil. Some differences appear to exist between insects from non-diapausing and diapausing colonies while no significant differences in community composition existed between the insects reared on different soils. Despite differences in the bacteria present in immature stages and in male and female adults, there is a possible core bacteriome of approximately 16 operational taxonomic units (i.e., present across all life stages). This research may provide insights into Bt resistance development, improved nutrition in artificial rearing systems, and new management strategies.

Author Correction: A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins.

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A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins.

Insect resistance to transgenic crops is a growing concern for farmers, regulatory agencies, the seed industry, and researchers. Since 2009, instances of field-evolved Bt resistance or cross resistance have been documented for each of the four Bt proteins available for western corn rootworm (WCR), a major insect pest. To characterize resistance, WCR populations causing unexpected damage to Bt maize are evaluated in plant and/or diet toxicity assays. Currently, it is not possible to make direct comparisons of data from different Bt proteins due to differing proprietary artificial diets. Our group has developed a new, publicly available diet (WCRMO-1) with improved nutrition for WCR larvae. For the current manuscript, we tested the compatibility of all Bt proteins currently marketed for WCR on the WCRMO-1 diet and specific proprietary diets corresponding to each toxin using a susceptible colony of WCR. We also tested WCR colonies selected for resistance to each protein to assess the ability of the diet toxicity assay to detect Bt resistance. The WCRMO-1 diet is compatible with each of the proteins and can differentiate resistant colonies from susceptible colonies for each protein. Our diet allows researchers to monitor resistance without the confounding nutritional differences present between diets.

Comparison of Six Artificial Diets for Western Corn Rootworm Bioassays and Rearing.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is considered the most important maize (Zea mays L.) pest in the U.S. Corn Belt. Bioassays testing susceptibility to Bacillus thuringiensis Berliner (Bt) and other toxins of corn rootworm larvae often rely on artificial diet formulations. Successful bioassays on artificial diet for corn rootworm have sometimes been challenging because of microbial contamination. Toward the long-term goal of developing a universal artificial diet for western corn rootworm larvae, we compared larval survival, dry weight, and percentage of molt in 10-d bioassays from six current diets of which we were aware. In addition, as part of longer term rearing efforts, we recorded molting over an extended period of development (60 d). Six different artificial diets, including four proprietary industry diets (A, B, C, and D), the first published artificial diet for western corn rootworm (Pleau), and a new diet (WCRMO-1) were evaluated. Western corn rootworm larval survival was above 90% and contamination was 0% on all diets for 10 d. Diet D resulted in the greatest dry weight and percentage molting when compared with the other diets. Although fourth-instar western corn rootworm larvae have not been documented previously (only three instars have been previously documented), as many as 10% of the larvae from Diet B molted into a fourth instar prior to pupating. Overall, significant differences were found among artificial diets currently used to screen western corn rootworm. In order for data from differing toxins to be compared, a single, reliable and high-quality western corn rootworm artificial diet should eventually be chosen by industry, academia, and the public as a standard for bioassays.

Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious insect pest of corn (Zea mays L.) in the United States and parts of Europe, and arguably one of the world's most expensive pests to control. Several diet formulations are currently used by industry and public researchers to evaluate WCR larvae in diet-toxicity bioassays. However, a publicly available diet that produces normative insects that are physiologically similar to WCR larvae reared on corn roots will accelerate development of management technologies. We report a new diet formulation that supports improved weight gain, larval development and survival compared with the only public diet for WCR that is currently available in the refereed literature. The formulation was created by using response surface methods combined with n-dimensional mixture designs to identify and improve the formulation of key ingredients. Weight gain increased two-fold, and survival and molting rates increased from 93% and 90%, respectively when reared on the public diet, to approximately 99% for both survival and molting at 11 days when reared on our new formulation. This new formulation provides a standardized growth medium for WCR larvae that will facilitate comparison of research results from various working groups and compliance with regulatory requirements.

Development and Characterization of MIR604 Resistance in a Western Corn Rootworm Population (Coleoptera: Chrysomelidae).

mCry3A is one of only four proteins licensed for commercial use in Diabrotica control. Utilizing a colony of western corn rootworm, Diabrotica virgifera virgifera LeConte, selected for resistance to mCry3A, we evaluated how mCry3A resistance was inherited and whether fitness costs were associated with mCry3A resistance. Reciprocal crosses between a selected colony and a control colony were performed; resulting progeny along with parent colonies were evaluated in dose toxicity assays, greenhouse assays, and seedling assays. Dose toxicity assay results were inconclusive, as the highest dose of protein tested did not produce sufficient mortality for accurate LC50 calculation. In whole-plant greenhouse assays on mCry3A-expressing corn, larval relative survival of the selected female × control male reciprocal cross was similar to that of the selected colony, while that of the control female × selected male was intermediate between the mCry3-selected colony and the control colony. However, when adult relative survival in whole-plant greenhouse assays was examined, no significant difference between the reciprocal crosses and the two parent colonies was detected. Heritability calculations based on both larval (0.66) and adult (1.03) survival data indicate that resistance to mCry3A is not inherited in a recessive manner. The selected colony was removed from selection pressure and evaluated after three or eight generations of removal. At three generations of removal from selection, a slight decrease in larval relative survival was detected compared with the selected colony. At eight generations of removal from selection, larval relative survival was comparable with that of the selected colony.

Multiple Assays Indicate Varying Levels of Cross Resistance in Cry3Bb1-Selected Field Populations of the Western Corn Rootworm to mCry3A, eCry3.1Ab, and Cry34/35Ab1.

Minnesota populations of Diabrotica virgifera virgifera LeConte, the western corn rootworm, surviving Cry3Bb1-expressing corn in the field and western corn rootworm populations assumed to be susceptible to all Bt proteins were evaluated for susceptibility to Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1 in diet assays and three different plant-based assays. Rootworm populations originating from Cry3Bb1 fields and that consistently experienced greater than expected damage had increased survival and larval growth compared to control populations assayed on Cry3Bb1 as well as mCry3a and eCry3.1Ab. Cross resistance was documented between Cry3Bb1 and both mCry3A and eCry3.1Ab as single toxins. Despite very high resistance ratios in some comparisons, cross resistance was not complete and also varied with the population being evaluated, the trait measured, and the susceptible rootworm population used for comparison. Regardless of resistance and cross resistance, all proteins, even Cry3Bb1, retained some efficacy in terms of either reducing rootworm larval growth, protecting plants from damage, or both, for all rootworm populations evaluated. For one Cry3Bb1-selected population, a resistance ratio of 9.1-fold was found to Cry34/35Ab1 when evaluating EC 50 values relative to a susceptible control population; however, resistance to Cry34/35Ab1 was not evident in all assays in this population. The United States Environmental Protection Agency recently suggested eliminating diet assays as part of the Bt resistance monitoring process. However, given the variability of responses of western corn rootworm populations to different proteins in different assays, both plant and diet assays are needed as options for detecting and fully characterizing resistance.

Aversion and attraction to harmful plant secondary compounds jointly shape the foraging ecology of a specialist herbivore.

Most herbivorous insect species are restricted to a narrow taxonomic range of host plant species. Herbivore species that feed on mustard plants and their relatives in the Brassicales have evolved highly efficient detoxification mechanisms that actually prevent toxic mustard oils from forming in the bodies of the animals. However, these mechanisms likely were not present during the initial stages of specialization on mustard plants ~100 million years ago. The herbivorous fly Scaptomyza nigrita (Drosophilidae) is a specialist on a single mustard species, bittercress (Cardamine cordifolia; Brassicaceae) and is in a fly lineage that evolved to feed on mustards only in the past 10-20 million years. In contrast to many mustard specialists, S. nigrita does not prevent formation of toxic breakdown products (mustard oils) arising from glucosinolates (GLS), the primary defensive compounds in mustard plants. Therefore, it is an appealing model for dissecting the early stages of host specialization. Because mustard oils actually form in the bodies of S. nigrita, we hypothesized that in lieu of a specialized detoxification mechanism, S. nigrita may mitigate exposure to high GLS levels within plant tissues using behavioral avoidance. Here, we report that jasmonic acid (JA) treatment increased GLS biosynthesis in bittercress, repelled adult female flies, and reduced larval growth. S. nigrita larval damage also induced foliar GLS, especially in apical leaves, which correspondingly displayed the least S. nigrita damage in controlled feeding trials and field surveys. Paradoxically, flies preferred to feed and oviposit on GLS-producing Arabidopsis thaliana despite larvae performing worse in these plants versus non-GLS-producing mutants. GLS may be feeding cues for S. nigrita despite their deterrent and defensive properties, which underscores the diverse relationship a mustard specialist has with its host when lacking a specialized means of mustard oil detoxification.

Near-isogenic lines for measuring phenotypic effects of DIMBOA-Glc methyltransferase activity in maize.

Three O-methyltransferases (BX10a, b, c) catalyze the conversion of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIM BOA-Glc) to 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA -Glc) in maize (Zea mays). Variation in benzoxazinoid accumulation and resistance to Rhopalosiphum maidis (corn leaf aphid) was attributed to a natural CACTA family transposon insertion that inactivates Bx10c. Whereas maize inbred line B73 has this transposon insertion, line CM L277 does not. To characterize the phenotypic effects of DIM BOA-Glc methyltransferase activity, we created near-isogenic lines derived from B73 and CM L277 that do or do not contain the transposon insertion. Bx10c inactivation causes high DIM BOA -Glc, low HDMBOA-Glc, and decreased aphid reproduction relative to near-isogenic lines that have a functional Bx10c gene. These results confirm the importance of this locus in maize aphid resistance. The availability of Bx10c near-isogenic lines will facilitate further research on the function of different benzoxazinoids and DIM BOA-Glc methyltransferase activity in maize defense against herbivores and pathogens.