Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.

Enhancing insecticidal efficacy of Bacillus thuringiensis Cry1Ab through pH-sensitive encapsulation.

Nanotechnology is a promising way to enhance the stability of Bacillus thuringiensis (Bt) insecticidal proteins under environmental conditions. In this work, two emulsions were prepared through the Pickering emulsion technique, stabilized by Cu2+-SQDs/S-CN nanocomposites and by GO nanosheets. In addition, a pH-sensitive polymer was incorporated into these emulsions, allowing the Bt protein, Cry1Ab, to be released in an alkaline pH environment, as it occurs in the lepidopteran pests' gut. The effectiveness of these two nanomaterials in protecting Cry1Ab from degradation, and therefore enhancing its pesticidal activity, was assessed by exposing samples of the purified unprotected protein and encapsulated protein to high-intensity UV light and 40°C temperature treatments. The UV treatment results were evaluated using SDS-PAGE analysis and pointed out that Cry1Ab could be structurally protected by the emulsions. The bioassays with first instar larvae of the lepidopteran pest Ostrinia nubilalis confirm the nanomaterial protection to UV and temperature treatments, i.e., decreasing about half the degradation rate and increasing up to 12-fold the residual activity after UV treatment. Our results indicate that encapsulation could be an effective strategy to improve the effectiveness of Cry1Ab under environmental conditions. KEY POINTS: • Pickering emulsions are effective for solubilized Cry1Ab encapsulation. • Structural and toxicity Cry1Ab properties are enhanced by pH-sensitive encapsulation. • Cu2+-SQDs/S-CN and GO nanomaterials improve the efficacy of Bt insecticides.

COMPILE: a GWAS computational pipeline for gene discovery in complex genomes.

BACKGROUND: Genome-Wide Association Studies (GWAS) are used to identify genes and alleles that contribute to quantitative traits in large and genetically diverse populations. However, traits with complex genetic architectures create an enormous computational load for discovery of candidate genes with acceptable statistical certainty. We developed a streamlined computational pipeline for GWAS (COMPILE) to accelerate identification and annotation of candidate maize genes associated with a quantitative trait, and then matches maize genes to their closest rice and Arabidopsis homologs by sequence similarity. RESULTS: COMPILE executed GWAS using a Mixed Linear Model that incorporated, without compression, recent advancements in population structure control, then linked significant Quantitative Trait Loci (QTL) to candidate genes and RNA regulatory elements contained in any genome. COMPILE was validated using published data to identify QTL associated with the traits of α-tocopherol biosynthesis and flowering time, and identified published candidate genes as well as additional genes and non-coding RNAs. We then applied COMPILE to 274 genotypes of the maize Goodman Association Panel to identify candidate loci contributing to resistance of maize stems to penetration by larvae of the European Corn Borer (Ostrinia nubilalis). Candidate genes included those that encode a gene of unknown function, WRKY and MYB-like transcriptional factors, receptor-kinase signaling, riboflavin synthesis, nucleotide-sugar interconversion, and prolyl hydroxylation. Expression of the gene of unknown function has been associated with pathogen stress in maize and in rice homologs closest in sequence identity. CONCLUSIONS: The relative speed of data analysis using COMPILE allowed comparison of population size and compression. Limitations in population size and diversity are major constraints for a trait and are not overcome by increasing marker density. COMPILE is customizable and is readily adaptable for application to species with robust genomic and proteome databases.

Gut Microbiota of Ostrinia nubilalis Larvae Degrade Maize Cellulose.

Most arthropod guts harbor diverse microbiota for symbiotic digestion. The European corn borer (ECB), Ostrinia nubilalis (Hübner), is a devastating pest that feeds the lignocellulose-rich tissues of maize plants. However, the potential role of ECB gut microbes in degrading maize cellulose remains largely unexplored. Here, we investigated the gut microbiota of ECB fed with different diets and their potential function in maize lignocellulose degradation. The diversity and composition of gut bacterial communities varied dramatically between the ECB larva fed with artificial diets (ECB-D) and maize plants (ECB-M). Draft genomes of the microbial consortia from ECB-D and ECB-M showed that the principal degraders of cellulose mainly belonged to Firmicutes or Proteobacteria and they were primarily found in the midgut. The cellulolytic microbial consortia contained genes encoding various carbohydrate-active enzymes (CAZyme). Furthermore, scanning electron microscopy revealed significant breakdown of lignocellulose in maize treated by the two microbial consortia for 9 days in vitro. Metabolomic analyses show that maize particles treated by two microbial consortia generate distinctive metabolomic profiles, with enrichment for different monosaccharides (i.e., Glucose, Rhamnofuranose, Isomaltose, and Cellobiose) and amino acids (i.e., Threonine, Histidine, and Lysine). The results indicated that the diet of the host impacted the composition and function of its gut microbiota and ECB exploited specific gut microbes to digest maize lignocellulose with distinctive products. Our study provides valuable microbiota resources for lignocellulose bioconversion.

Concerted cis and trans effects underpin heightened defense gene expression in multi-herbivore-resistant maize lines.

In maize (Zea mays ssp. mays), agriculturally damaging herbivores include lepidopteran insects, such as the European corn borer (Ostrinia nubilalis), and distantly related arthropods, like the two-spotted spider mite (Tetranychus urticae). A small number of maize lines, including B96 and B75, are highly resistant to both herbivores, and B96 is also resistant to thrips. Using T. urticae as a representative pest that causes significant leaf tissue damage, we examined the gene expression responses of these lines to herbivory in comparison with each other and with the susceptible line B73. Upon herbivory, the most resistant line, B96, showed the strongest gene expression response, with a dramatic upregulation of genes associated with jasmonic acid biosynthesis and signaling, as well as the biosynthesis of specialized herbivore deterrent compounds, such as death acids and benzoxazinoids. Extending this work with allele-specific expression analyses in F1 hybrids, we inferred that the concerted upregulation of many defense genes, including the majority of benzoxazinoid biosynthetic genes in B96, as compared with B73, for the herbivore treatment, resulted from an assemblage of trans control and multiple cis effects acting with similar directionality on gene expression. Further, at the level of individual and potentially rate limiting genes in several major defense pathways, cis and trans effects acted in a reinforcing manner to result in exceptionally high expression in B96. Our study provides a comprehensive resource of cis elements for maize lines important in breeding efforts for herbivore resistance, and reveals potential genetic underpinnings of the origins of multi-herbivore resistance in plant populations.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

An agent-based model of insect resistance management and mitigation for Bt maize: a social science perspective.

BACKGROUND: Farmers around the world have used Bt maize for more than two decades, delaying resistance using a high-dose/refuge strategy. Nevertheless, field-evolved resistance to Bacillus thuringiensis (Bt) toxins has been documented. This paper describes a spatially explicit population genetics model of resistance to Bt toxins by the insect Ostrinia nubilalis and an agent-based model of farmer adoption of Bt maize incorporating social networks. The model was used to evaluate multiple resistance mitigation policies, including combinations of increased refuges for all farms, localized bans on Bt maize where resistance develops, area-wide sprays of insecticides on fields with resistance and taxes on Bt maize seed for all farms. Evaluation metrics included resistance allele frequency, pest population density, farmer adoption of Bt maize and economic surplus. RESULTS: The most effective mitigation policies for maintaining a low resistance allele frequency were 50% refuge and localized bans. Area-wide sprays were the most effective for maintaining low pest populations. Based on economic surplus, refuge requirements were the recommended policy for mitigating resistance to high-dose Bt maize. Social networks further enhanced the benefits of refuges relative to other mitigation policies but accelerated the emergence of resistance. CONCLUSION: These results support using refuges as the foundation of resistance mitigation for high-dose Bt maize, just as for resistance management. Other mitigation policies examined were more effective but more costly. Social factors had substantial effects on the recommended management and mitigation of insect resistance, suggesting that agent-based models can make useful contributions for policy analysis.

Conventional Soil Management May Promote Nutrients That Lure an Insect Pest to a Toxic Crop.

Slow and consistent nutrient release by organic fertilizers can improve plant nutrient balance and defenses, leading to herbivore avoidance of organically managed crops in favor of conventional crops with weaker defenses. We propose that this relative attraction to conventional plants, coupled with the use of genetically modified, insecticidal crops (Bt), has created an unintentional attract-and-kill system. We sought to determine whether Bt and non-Bt corn Zea mays L. plants grown in soil collected from five paired organic and conventional fields differed in attractiveness to European corn borer [Ostrinia nubilalis (Hübner)] moths, by conducting ovipositional choice and flight tunnel assays. We then examined the mechanisms driving the observed differences in attraction by comparing soil nutrient profiles, soil microbial activity, plant nutrition, and plant volatile profiles. Finally, we assessed whether European corn borer abundance near corn fields differed based on soil management. European corn borer preferred plants grown in conventional soil but did not discriminate between Bt and non-Bt corn. Organic management and more alkaline soil were associated with an increased soil magnesium:potassium ratio, which increased plant magnesium, and were linked to reduced European corn borer oviposition. There was an inconsistent trend for higher European corn borer moth activity near conventional fields. Our results extend the mineral balance hypothesis describing conventional plant preference by showing that it can also improve attraction to plants with genetically inserted toxins. Unintentional attract (to conventional) and (Bt) kill is a plausible scenario for pest declines in response to Bt corn adoption, but this effect may be obscured by variation in other management practices and landscape characteristics.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis.

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis.

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae.

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is dependent upon the stability of double-stranded RNA (dsRNA) molecules. In many insects, especially lepidopteran species, RNAi efficiency is limited by high instability of dsRNA in the gut and/or hemolymph, preventing the development of RNAi-based strategies for many serious pests. Previous attempts to perform RNAi on Ostrinia nubilalis (ECB, Lepidoptera: Crambidae) indicate low RNAi efficiency with both dsRNA injection and feeding. To investigate the contribution of dsRNA instability to low RNAi efficiency in ECB, a serious of ex vivo incubation experiments were performed where dsRNA integrity was assessed following incubation in larval gut continents and hemolymph using gel electrophoresis or RT-qPCR. DsRNA was less stable in the gut contents from ECB than in gut contents from Diabrotica virgifera virgifera, a coleopteran exhibiting high RNAi efficiency. Furthermore, characterization of dsRNA stability in ECB gut contents and hemolymph revealed that dsRNA was rapidly degraded under physiologically relevant conditions as a result of enzymatic activity that was neither size- nor sequence-dependent. These findings suggest that instability of dsRNA in ECB tissues is a contributing factor to the poor efficiency of RNAi in this pest. This work advances our understanding of mechanisms impacting RNAi efficiency in ECB and related lepidopteran insects for which novel pest management strategies are needed, and may facilitate the development of strategies for enhancing dsRNA stability in ECB tissues.

Using gyroplane for application of Trichogramma spp. against the European corn borer in maize.

BACKGROUND: Achieving sustainability of agricultural production is one of the key challenges for humanity. Minimizing the use of chemicals and replacing them with biological plant protection is, firstly, fully in line with the agriculture's development strategy and, secondly, confirmed to be achievable in practice. For instance, many researchers point to the potential of using Trichogramma spp. to control Ostrinia nubilalis in maize. However, the application methods for preparations containing Trichogramma spp. are still at the stage of development. So far, ground application proved time and energy consuming. Aerial application using ultralight aircraft is a new option. RESULTS: The present study presents the results of tests on the effectiveness of applying Trichogramma spp. from the board of an ultralight gyroplane. A 6-year experiment was conducted on a maize monoculture located in south-western Poland. The results indicate that the low-height aerial application allows precise dosing and satisfactory distribution of the biopesticide. The efficacy of 60-85% (depending on the year) of the gyroplane-based spraying operations was comparable with that observed for ground application. CONCLUSION: Considering the speed and high efficiency of the treatment, using a gyroplane as a carrier of the biopesticide application system is a good alternative to the other methods. © 2020 Society of Chemical Industry.

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.

Comparisons of Transcriptional Profiles of Gut Genes between Cry1Ab-Resistant and Susceptible Strains of Ostrinia nubilalis Revealed Genes Possibly Related to the Adaptation of Resistant Larvae to Transgenic Cry1Ab Corn.

A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.

Can the Use of Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) to Control Ostrinia nubilalis (Lepidoptera: Crambidae) Be Economically Sustainable for Processing Sweet Corn?

European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is the main pest causing damage to sweet corn in North America. Conventional management with multiple use of insecticides is a common practice for processing sweet corn. In Canada, the use of Trichogramma spp. began in the 1990s, but the adoption of this approach for European corn borer management is still limited to the fresh market of sweet corn. Trichogramma ostriniae (Peng & Chen) has great potential as a biological control agent for large areas such as in processing sweet corn. The objective of this study was to evaluate an economically and environmentally sustainable alternative to insecticides for controlling European corn borer populations in processing sweet corn. During the growing season, the mean number of larvae decreased after insecticide (0.07 ± 0.04) and Trichogramma (1.32 ± 0.59) treatments compared with the control (2.42 ± 0.72). At harvest, damages associated with European corn borer were similar after Trichogramma (1.0 ± 0.7%) and insecticide (1.0 ± 0.6%) treatments, but significantly lower than the control (8.7 ± 3.3%). This study showed that the use of T. ostriniae can significantly decrease the pressure exerted by European corn borer and its damage on corn ears. This outcome is particularly interesting considering that it was achieved with lower doses of Trichogramma, a lower number of releases, and on large crop areas, compared with what is actually done to protect fresh market corn from European corn borer. Under these conditions, the use of Trichogramma is an economically and competitive alternative to insecticide applications.

Optimizing the Point-Source Emission Rates and Geometries of Pheromone Mating Disruption Mega-Dispensers.

High-emission-rate "mega-dispensers" have come into increasing use for sex pheromone mating disruption of moth pests over the past two decades. These commercially available dispensers successfully suppress mating and reduce crop damage when they are deployed at very low to moderate densities, ranging from 1 to 5/ha to 100-1000/ha, depending on the dispenser types and their corresponding pheromone emission rates. Whereas traditionally the emission rates for successful commercial mating disruption formulations have been measured in terms of amounts (usually milligram) emitted by the disruptant application per acre or hectare per day, we suggest that emission rates should be measured on a per-dispenser per-minute basis. In addition we suggest, because of our knowledge concerning upwind flight of male moths being dependent on contact with pheromone plume strands, that more attention needs to be paid to optimizing the flux within plume strands that shear off of any mating disruption dispenser's surface. By measuring the emission rates on a per-minute basis and measuring the plume strand concentrations emanating from the dispensers, it may help improve the ability of the dispensers to initiate upwind flight from males and initiate their habituation to the pheromone farther downwind than can otherwise be achieved. In addition, by optimizing plume strand flux by paying attention to the geometries and compactness of mating disruption mega-dispensers may help reduce the cost of mega-dispenser disruption formulations by improving their behavioral efficacy while maintaining field longevity and using lower loading rates per dispenser.

High regional genetic diversity and lack of host-specificity in Ostrinia nubilalis (Lepidoptera: Crambidae) as revealed by mtDNA variation.

The European corn borer (Ostrinia nubilalis) infests a wide array of host plants and is considered one of the most serious pests of maize in Europe. Recent studies suggest that individuals feeding on maize in Europe should be referred to O. nubilalis (sensu nov.), while those infesting dicots as Ostrinia scapulalis (sensu nov.). We test if the clear genetic distinctiveness among individuals of O. nubilalis living on maize vs. dicots is tracked by mitochondrial DNA (mtDNA). We used fragments of COI and COII genes of 32 individuals traditionally recognized as O. nubilalis collected on three host plants, maize, mugwort and hop, growing in different parts of Poland. In addition, we reconstructed the mtDNA phylogeny of Ostrinia species based on our data and sequences retrieved from GenBank to assess host and/or biogeographic patterns. We also compared haplotype variation found in Poland (east-central Europe) with other regions (Anatolia, Eastern Europe, Balkans, Far East, North America). Our study showed high mtDNA diversity of O. nubilalis in Poland in comparison with other regions and revealed rare haplotypes likely of Asian origin. We did not find distinct mtDNA haplotypes in larvae feeding on maize vs. dicotyledonous plants. Phylogenetic analyses showed an apparent lack of mtDNA divergence among putatively distinct lineages belonging to the O. nubilalis group as identical haplotypes are shared by Asian and European individuals. We argue that human-mediated dispersal, hybridization and sporadic host jumps are likely responsible for the lack of a geographic pattern in mtDNA variation.

On-farm evaluation of inundative biological control of Ostrinia nubilalis (Lepidoptera: Crambidae) by Trichogramma brassicae (Hymenoptera: Trichogrammatidae) in three European maize-producing regions.

BACKGROUND: A 2 year study was conducted to evaluate the efficacy of biological control with optimally timed Trichogramma brassicae releases as an integrated pest management tool against the European corn borer (ECB), Ostrinia nubilalis (Hübner), in on-farm experiments (i.e. real field conditions) in three European regions with dissimilar geoclimatic conditions and ECB pressure and conventional management (i.e. insecticide treated and untreated). RESULTS: Biological control with Trichogramma (1) provided ECB protection comparable with conventional management, (2) in all cases maintained mycotoxin levels below the EU threshold for maize raw materials destined for food products, (3) was economically sustainable in southern France and northern Italy, but not in Slovenia where it resulted in a significant decrease in gross margin, mainly owing to the cost of Trichogramma product, and (4) enabled avoidance of detrimental environmental effects of lambda-cyhalothrin use in northern Italy. CONCLUSION: Optimally timed mass release of T. brassicae could be considered a sustainable tool for IPM programmes against ECB in southern France and northern Italy. Better involvement of regional advisory services is needed for the successful dissemination and implementation of biological control. Subsidy schemes could also motivate farmers to adopt this IPM tool and compensate for high costs of Trichogramma product.

A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops.

Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet.