microRNA profiling between Bacillus thuringiensis Cry1Ab-susceptible and -resistant European corn borer, Ostrinia nubilalis (Hübner).

Transgenic maize hybrids that express insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins effectively protect against the European corn borer, Ostrinia nubilalis, a devastating maize pest. Field monitoring and laboratory selections have detected varying levels of O. nubilalis resistance to Cry1Ab toxin. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in post-transcriptional gene regulation. Their potential roles in the evolution of Bt resistance, however, remain largely unknown. Sequencing of small RNA libraries from the midgut of Cry1Ab-susceptible and resistant O. nubilalis larvae resulted in the discovery of 277 miRNAs, including 248 conserved and 29 novel. Comparative analyses of miRNA expression profiles between the laboratory strains predicted 26 and nine significantly up- and down-regulated transcripts, respectively, in the midgut of Cry1Ab resistant larvae. Amongst 15 differentially regulated miRNAs examined by quantitative real-time PCR, nine (60%) were validated as cosegregating with Cry1Ab resistance in a backcross progeny. Differentially expressed miRNAs were predicted to affect transcripts involved in cell membrane components with functions in metabolism and binding, and the putative Bt-resistance genes aminopeptidase N and cadherin. These results lay the foundation for future investigation of the potential role of miRNAs in the evolution of Bt resistance.

Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Inheritance of Cry1F resistance, cross-resistance and frequency of resistant alleles in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Transgenic maize, Zea maize L., expressing the Cry1F protein from Bacillus thuringiensis has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003. Unexpected damage to Cry1F maize was reported in 2006 in Puerto Rico and Cry1F resistance in S. frugiperda was documented. The inheritance of Cry1F resistance was characterized in a S. frugiperda resistant strain originating from Puerto Rico, which displayed >289-fold resistance to purified Cry1F. Concentration-response bioassays of reciprocal crosses of resistant and susceptible parental populations indicated that resistance is recessive and autosomal. Bioassays of the backcross of the F1 generation crossed with the resistant parental strain suggest that a single locus is responsible for resistance. In addition, cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry2Aa and Vip3Aa was assessed in the Cry1F-resistant strain. There was no significant cross-resistance to Cry1Aa, Cry1Ba and Cry2Aa, although only limited effects were observed in the susceptible strain. Vip3Aa was highly effective against susceptible and resistant insects indicating no cross-resistance with Cry1F. In contrast, low levels of cross-resistance were observed for both Cry1Ab and Cry1Ac. Because the resistance is recessive and conferred by a single locus, an F1 screening assay was used to measure the frequency of Cry1F-resistant alleles from populations of Florida and Texas in 2010 and 2011. A total frequency of resistant alleles of 0.13 and 0.02 was found for Florida and Texas populations, respectively, indicating resistant alleles could be found in US populations, although there have been no reports of reduced efficacy of Cry1F-expressing plants.

Role of a γ-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides.

The western corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was controlled by applications of cyclodiene insecticides from the late 1940s until resistance evolved ∼10 years later. Range expansion from the western plains into eastern USA coincides with resistance development. An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. We found that the non-synonymous single nucleotide polymorphism (SNP) G/T at the GABA receptor cDNA position 838 (G/T(838)) of D. v. virgifera resulted in the alanine to serine change, and the codominant SNP allele T(838) was genetically linked to survival of beetles in aldrin bioassays. A phenotypic gradient of decreasing susceptibility from west to east was correlated with higher frequencies of the resistance-conferring T(838) allele in the eastern-most populations. This pattern exists in opposition to perceived selective pressures since the more eastern and most resistant populations probably experienced reduced exposure. The reasons for the observed distribution are uncertain, but historical records of the range expansion combined with the distribution of susceptible and resistant phenotypes and genotypes provide an opportunity to better understand factors affecting the species' range expansion.

Effects of thiamethoxam seed treatments on soybean aphid (Hemiptera: Aphididae) feeding behavior.

Since its discovery in North America in 2000, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), has rapidly become an important pest of soybean [Glycine max (L.) Merrill], sometimes resulting in significant yield losses. Previous research has documented the toxicity of neonicotinoid seed treatments to soybean aphids, but control under field conditions has been inconsistent. Imidacloprid, a popular neonicotinoid insecticide, has been shown to exhibit antifeedant effects on aphids. Antifeedant activity has not been demonstrated for other neonicotinoids, including thiamethoxam. This research investigated the effects of a thiamethoxam seed treatment on soybean aphid feeding behavior by using electronic penetration graphs (EPG) to visualize stylet penetration behavior. Soybean aphid feeding behavior was assessed for 9 h on thiamethoxam-treated and untreated soybeans (V2 and V4 stages). Because results were inconclusive from initial experiments, a study was conducted to document the effects of thiamethoxam-treated soybeans on soybean aphid survival. The seed treatment was shown to negatively affect aphid survival at 4, 8, and 11 d after aphid introduction. A subsequent EPG study then was designed to document soybean aphid feeding behavior for 15 h, after an initial exposure of 9 h to thiamethoxam-treated soybeans. In this study, the exposed aphids exhibited significant differences in feeding behavior compared with those aphids feeding on untreated soybeans. Soybean aphids on thiamethoxam-treated soybeans spent significantly less time feeding in the sieve element phase, with a greater duration of nonprobing events. These studies suggest soybean aphids are unable to ingest phloem sap, which may be another important element in seed treatment protection.

Dose-response relationships of clothianidin, imidacloprid, and thiamethoxam to Blissus occiduus (Hemiptera: Blissidae).

The western chinch bug, Blissus occiduus Barber (Hemiptera: Blissidae), has emerged as a serious pest of buffalograss, Buchlod dactyloides (Nuttall) Engelmann. In general, neonicotinoid insecticides effectively control a variety of turfgrass insects, particularly phloem-feeding pests. However, because of well documented inconsistencies in control, these compounds are generally not recommended for chinch bugs. This study was designed to document the contact and systemic toxicity of three neonicotinoid insecticides (clothianidin, imidacloprid, and thiamethoxam) to B. occiduus. In contact bioassays, thiamethoxam was approximately 20-fold less toxic than clothianidin or imidacloprid to B. occiduus nymphs and three-fold more toxic to adults. In adult systemic bioassays, thiamethoxam was up to five-fold more toxic than clothianidin or imidacloprid. Interestingly, thiamethoxam was significantly more toxic to adults than to nymphs in both contact and systemic bioassays. This was not observed with clothianidin or imidacloprid. Bifenthrin, used for comparative purposes, exhibited 1844-fold and 122-fold increase in toxicity to nymphs and adults, respectively. These results provide the first documentation of the relative toxicity of these neonicotinoid insecticides to B. occiduus.

Bacillus thuringiensis resistance influences European corn borer (Lepidoptera: Crambidae) larval behavior after exposure to Cry1Ab.

The behavior of pests targeted by Bacillus thuringiensis (Bt) crops has been recognized as an important factor to define resistance management plans. However, most data do not include the possible impact resistance may have on the behavior of pests. To examine whether resistance influences behavior of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), neonates after exposure to dietary Bt, the responses of Cry1Ab-resistant, -susceptible, and hybrid (F1) lines from two populations were compared in laboratory tests by using artificial diet mixed with 10-50% Cry1Ab or non-Bt isoline corn, Zea mays L., tissue. In no-choice tests, resistant (and usually hybrid) lines were less likely to be irritated (i.e., to move away after physical contact with diet containing Cry1Ab) than susceptible larvae after exposure to diets containing 10-50% Cry1Ab leaf tissue. Early in the no-choice tests (8 h), neonate O. nubilalis also were more likely to move off of diets that contained 10% non-Bt tissue compared with diets with 25 or 50% non-Bt tissue. In agreement with results from no-choice tests, choice tests with 10 or 25% tissue indicated that resistant (and sometimes hybrid) larvae were more likely than susceptible neonates to be found on diet with Cry1Ab. For choice tests, differences among lines seemed dependent on the amount of Cry1Ab tissue incorporated into diets. Results suggest differences in behavior are a result of reduced physiological susceptibility to Cry1Ab and are not an independent behavioral component to resistance.

Inheritance of Cry1F resistance in laboratory-selected European corn borer and its survival on transgenic corn expressing the Cry1F toxin.

A major assumption of the high-dose/refuge strategy proposed for insect resistance management strategies for transgenic crop plants that express toxins from Bacillus thuringiensis is that resistance traits that evolve in pest species will be recessive. The inheritance of Cry1F resistance and larval survival on commercially available Cry1F corn hybrids were determined in a laboratory-selected strain of European corn borer, Ostrinia nubilalis (Hübner), displaying more than 3000-fold resistance to Cry1F. Concentration-response bioassays of reciprocal parental crosses indicated that the resistance is autosomal and recessive. Bioassays of the backcross of the F1 generation with the selected strain were consistent with the hypothesis that a single locus, or a set of tightly linked loci, is responsible for the resistance. Greenhouse experiments with Cry1F-expressing corn hybrids indicated that some resistant larvae survived the high dose of toxin delivered by Cry1F-expressing plants although F1 progeny of susceptible by resistant crosses had fitness close to zero. These results provide the first direct evidence that the high dose/refuge strategy currently in place to manage resistance in Cry1F-expressing corn is appropriate.

A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue.

A cadherin-like gene associated with larval midgut tissues was cloned from western corn rootworm (Diabrotica virgifera virgifera: Coleoptera), an economically important agricultural pest in North America and Europe and the primary target pest species for corn hybrids expressing Cry3 toxins from Bacillus thuringiensis (Bt). The full-length cDNA (5371 bp in length) encodes an open reading frame for a 1688 amino acid polypeptide. The putative protein has similar architecture to cadherin-like proteins isolated from lepidopteran midguts that have been shown to bind to Cry1 Bt toxins and have been implicated in Bt resistance. The D. v. virgifera cadherin-like gene is expressed primarily in the larval midgut and regulated during development, with high levels of expression observed in all instars and adults but not pupae. The corresponding genomic sequence spans more than 90 kb and is interspersed with 30 large introns. The genomic organization of the cadherin-like gene for this coleopteran species bears strong resemblance to lepidopteran cadherins suggesting a common molecular basis for susceptibility to Cry3 toxins in Coleoptera.

Inheritance of resistance to the Cry1Ab Bacillus thuringiensis toxin in Ostrinia nubilalis (Lepidoptera: Crambidae).

Laboratory selection with Cry1Ab, the predominant Bacillus thuringiensis (Bt) toxin in transgenic corn, Zea mays L., produced >1000-fold resistance in two laboratory strains of European corn borer, Ostrinia nubilalis (Hübner). We tested the offspring of various crosses to determine the mode of inheritance of resistance to Cry1Ab. Patterns of inheritance of resistance were similar in the two resistant strains. The progeny of reciprocal F1 crosses (resistant male x susceptible female and vice versa) responded alike in bioassays, indicating autosomal inheritance. The median lethal concentrations (LC50 values) of F1 were intermediate between the resistant and susceptible parents, indicating approximately additive inheritance. However, the dominance of resistance increased as the concentration of Cry1Ab decreased. Analysis of progeny from backcrosses (F1 x susceptible strain) suggests that resistance was controlled by more than one locus. In particular, the fit of observed to expected mortality improved as the number of putative loci increased from 1 to 10. The polygenic nature of resistance in these two laboratory strains suggests that major genes for resistance to Cry1Ab were not common in the founding populations of O. nubilalis. A low initial frequency of major genes for Cry1Ab resistance might be an important factor in delaying evolution of resistance to Bt corn in this pest.

Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen.

Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

Impact of Bt corn pollen on monarch butterfly populations: a risk assessment.

A collaborative research effort by scientists in several states and in Canada has produced information to develop a formal risk assessment of the impact of Bt corn on monarch butterfly (Danaus plexippus) populations. Information was sought on the acute toxic effects of Bt corn pollen and the degree to which monarch larvae would be exposed to toxic amounts of Bt pollen on its host plant, the common milkweed, Asclepias syriaca, found in and around cornfields. Expression of Cry proteins, the active toxicant found in Bt corn tissues, differed among hybrids, and especially so in the concentrations found in pollen of different events. In most commercial hybrids, Bt expression in pollen is low, and laboratory and field studies show no acute toxic effects at any pollen density that would be encountered in the field. Other factors mitigating exposure of larvae include the variable and limited overlap between pollen shed and larval activity periods, the fact that only a portion of the monarch population utilizes milkweed stands in and near cornfields, and the current adoption rate of Bt corn at 19% of North American corn-growing areas. This 2-year study suggests that the impact of Bt corn pollen from current commercial hybrids on monarch butterfly populations is negligible.

Expression and induction of three family 4 cytochrome P450 (CYP4)* genes identified from insecticide-resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera.

We have previously determined that cytochrome P450-based oxidation is involved in resistance to the insecticides methyl parathion and carbaryl in geographically distinct Nebraska western corn rootworm populations. Three new family 4 cytochrome P450 (CYP4) gene fragments (CYP4AJ1, CYP4G18 and CYP4AK1) were cloned and sequenced from insecticide-resistant and -susceptible western corn rootworms. Insecticide bioassays indicated the resistant population employed in this study was significantly resistant to the insecticides methyl parathion and carbaryl. CYP4AJ1 and CYP4G18 were cloned from both genomic PCR and RT-PCR products, although only CYP4AJ1 contains an intronic region. Alignments of inferred amino acid sequences with other homologous insect CYP4 genes indicates a high degree of similarity. Northern analysis concurrently employing mixed probes representing each of the three rootworm CYP4 fragments identified increased mRNA transcript signals (i) in resistant rootworms and (ii) following induction by the P450 inducer pentamethyl benzene. These results support our previous documentation of P450-based insecticide resistance and suggest increased CYP4 transcript abundance can serve as a molecular resistance-associated marker.

Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis toxin.

Transgenic corn expressing the insecticidal toxin from Bacillus thuringiensis Berliner is gaining support as an effective control technology for use against lepidopteran pests, particularly European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). However, there is concern that widespread adoption of transgenic plants will rapidly lead to B. thuringiensis toxin resistance. Thus, long-term selection of O. nubilalis populations with the Cry1Ab B. thuringiensis toxin has been undertaken in several laboratories in the United States and in Europe. We present results from two independent selection experiments performed in laboratories at the University of Nebraska and at the Institut National de la Recherche Agronomique in France. Although the protocols and methods used by the two laboratories were different, the results were comparable. The highest level of resistance occurred at generation 7 (14-fold), generation 9 (13-fold), and generation 9 (32-fold) for three different strains. For each strain, the level of resistance fluctuated from generation to generation, although there were consistently significant decreases in toxin susceptibility across generations for all selected strains. These results suggest that low levels of resistance are common among widely distributed O. nubilalis populations.

Baseline susceptibility of the corn earworm (Lepidoptera: Noctuidae) to the Cry1Ab toxin from Bacillus thuringiensis.

Susceptibility to Cry1Ab toxin from Bacillus thuringiensis (Bt) was determined for 12 field populations of neonate corn earworm, Helicoverpa zea (Boddie), from the United States. Earworm larvae were exposed to artificial diet treated with increasing Bt concentrations, and mortality and growth inhibition were evaluated after 7 d. The range of variation in Bt susceptibility indicated by growth inhibition was very similar to that indicated by mortality. Although interpopulation variation in susceptibility to both proteins was observed, the magnitude of the differences was small (less than or equal to fivefold). These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among corn earworm populations rather than variation caused by prior exposure to selection pressures. Therefore, corn earworms apparently are susceptible to Bt toxins across most of their geographic range.

Development of diagnostic concentrations for monitoring Bacillus thuringiensis resistance in European corn borer (Lepidoptera: Crambidae).

Two candidate diagnostic concentrations of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis corresponding to the LC99 and EC99 (effective concentration that causes 99% growth inhibition) for European corn borer, Ostrinia nubilalis (Hübner), were determined based on previously obtained baseline data. Validation experiments using field-collected European corn borer populations from across North America showed that for Cry1Ab, a concentration corresponding to the upper limit of the 95% confidence interval of the LC99, produced mortality > 99% for all populations tested. However, for Cry1Ac, adjustments and further validation are probably necessary. Development of B. thuringiensis resistance monitoring programs that rely on diagnostic techniques are discussed.

Atrazine induction of cytochrome P450 in Chironomus tentans larvae.

Cytochrome P450-dependent aldrin epoxidation was characterized in third instar larvae of the aquatic midge, Chironomus tentans. Optimal in vitro assay conditions for the epoxidase were pH 7.6 and 31 degrees C. Activity was linear up to 40 min of incubation time and 0.5 mg microsomal protein per incubation. The activity was concentrated in the microsomal fraction of whole body homogenates and was NADPH-dependent. The effect of atrazine exposure on aldrin epoxidase was measured to determine if this herbicide induces cytochrome P450-dependent activity. Comparisons of control and atrazine-exposed midges indicated increased epoxidase activity as a result of atrazine exposure, and a 45 kDa protein of increased intensity was observed after SDS-PAGE of microsomal protein. The molecular weight of this protein was similar in size to cytochrome P450 enzymes reported for other insects. Heme staining of SDS-PAGE gels and immunochemical studies using a Drosophila melanogaster anti-P450 polyclonal antiserum, further support the cytochrome P450 nature of this inducible 45 kDa protein.

Larval susceptibility of an insecticide-resistant western corn rootworm (Coleoptera: Chrysomelidae) population to soil insecticides: laboratory bioassays, assays of detoxification enzymes, and field performance.

Soil insecticides were evaluated in laboratory and field studies against larvae of an insecticide resistant population (Phelps County, NE) of western corn rootworm, Diabrotica virgifera virgifera LeConte. Insecticide toxicity was evaluated by topical application of technical insecticides to 3rd instars from Saunders County, NE (susceptible) and Phelps County populations. Resistance ratios (LD50 Phelps County/LD50 Saunders County) for the insecticides methyl parathion, tefluthrin, carbofuran, terbufos, and chlorpyrifos were 28.0, 9.3, 8.7, 2.6 and 1.3, respectively. Biochemical investigation of suspected enzymatic resistance mechanisms in 3rd instars identified significant elevation of esterase activity (alpha and beta naphthyl acetate hydrolysis [3.8- and 3.9-fold]). Examination of 3rd instar esterases by native PAGE identified increased intensity of several isoenzymes in the resistant population. Assays of cytochrome P450 activity (4-CNMA demethylation and aldrin epoxidation) did not identify elevated activity in resistant 3rd instars. Granular soil insecticides were applied at planting to corn, Zea mays L., in replicated field trials in 1997 and 1998 at the same Phelps County site as the source of resistant rootworms for the laboratory studies. In 1997, planting time applications of Counter 20CR, Counter 15 G (terbufos), and Lorsban 15 G (chlorpyrifos) resulted in the lowest root injury ratings (1-6 Iowa scale); 2.50, 2.55, 2.65, respectively (untreated check root rating of 4.55). In 1998, all insecticides performed similarly against a lower rootworm density (untreated check root rating of 3.72). These studies suggest that resistance previously documented in adults also is present in 3rd instars, esterases are possibly involved as resistance mechanisms, and resistance to methyl parathion in adults is also evident in larvae, but does not confer cross-resistance in larvae to all organophosphate insecticides.