Feeding behavior of neonate Ostrinia nubilalis (Lepidoptera: Crambidae) on Cry1Ab Bt corn: implications for resistance management

The European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is an economically important insect pest of corn, Zea mays L., in the United States and Canada. The development of genetically modified corn expressing genes derived from Bacillus thuringiensis (Bt) that encodes insecticidal crystalline (Cry) proteins has proven to be effective in controlling this insect. To assess the feeding behavior of neonate O. nubilalis on Bt corn, we examined differences in feeding behavior, based on presence of plant material in the gut, between Cry1Ab Bt corn and non-Bt near isoline corn for four intervals over a 48-h period. Feeding experiments revealed that there was significantly less feeding on Bt corn compared with non-Bt near isoline corn. The behavior of neonates on the plant corresponded with the differences in feeding on the two corn lines. The findings also showed that > 50% of the larvae initially left the plant before there was evidence in the gut of feeding regardless of whether the source was Bt or non-Bt corn. A higher quantity of plant material was found in the gut of larvae recovered from leaves of non-Bt compared with Bt corn. At the end of 48 h among the larvae that had left the plant, a greater proportion from Bt corn had plant material in the gut than did those from non-Bt corn.     

Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of the European corn borer,Ostrinia nubilalis

Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis ( Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)). We used an F(2) screen on >1,200 isofemale lines of Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80%. In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.     

Peritrophic membrane structure and secretion in European corn borer larvae (Ostrinia nubilalis)

Peritrophic membrane (PM) secretion and formation occur primarily in the anterior region of the mesenteron in the European corn borer (Ostrinia nubilalis) as determined by light and electron microscopy. Nascent PM first became visible as fibrous linear chitin-containing structures stained with gold-labeled wheat germ agglutinin between and at the tips of the microvilli. No formed PM was visible at the foregut-midgut junction, but a thin single PM appeared first in the lumen between the stomodeal valves and the midgut epithelium. Just posterior to the stomodeal valves, multiple PMs were observed that became progressively thicker and more numerous in the mid and posterior regions of the mesenteron. The PM consists of an orthogonal chitin meshwork with openings slightly larger than the diameters of the microvilli. As it delaminates from the microvilli, the meshwork becomes embedded in proteinaceous matrix that greatly reduces the pore size of the PM.     

Evaluation of Ostrinia nubilalis (Lepidoptera: Crambidae) neonate preferences for corn and weeds in corn

Choice tests were conducted to determine feeding preferences of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), neonates for 15 species of plants. Percentage of neonates accepting (found on) each leaf disc after 24 h was measured using choice tests. Initially, nine species of plants were evaluated. The following year, 10 plant species were evaluated during O. nubilalis first generation and 11 species during the second generation. Pennsylvania smartweed, Polygonum pennsylvanicum (L.), had the highest percentage of neonates accepting leaf discs in both years. Other plants with high acceptance rates included swamp smartweed, Polygonum amphibium L.; velvetleaf, Abutilon theophrasti Medicus; cocklebur, Xanthium strumarium L.; and yellow foxtail, Setaria glauca (L.). Corn, Zea mays L., consistently had low percentages of neonates accepting leaf discs along with common waterhemp, Amaranthus rudis Sauer. Implications these results may have on O. nubilalis host plant selection in central Iowa's corn dominated landscape are considered.     

Evaluating resistance to Bt toxin Cry1Ab by F2 screen in European populations of Ostrinia nubilalis (Lepidoptera: Crambidae)

The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the Cry1Ab toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the Cry1Ab toxin. The cost of F2 screen performed in the four countries varied from U.S. dollars 300 to dollars 1300 per line screened. The major difference in cost was mostly due to a severe loss of univoltine lines during the screen in Germany and Slovakia. In none of the screened lines did we detect alleles conferring resistance to Mon810 maize or to the Cry1Ab toxin. The frequency of resistance alleles were < 1.0 x 10(-3), < 1.6 x 10(-3), < 9.2 x 10(-3), and < 2.6 x 10(-2) in France, Germany, Italy, and Slovakia, with 95% probability, respectively. The average detection probability over all lines was approximately 90%. Making the assumption that European corn borer populations in these countries belong to the same genetic entity, the frequency of alleles conferring resistance to the Cry1Ab produced by the Mon810 maize in western and central Europe was 1.0 x 10(-4), with a 95% confidence interval of 0-3.0 x 10(-4).     

Isolation and inhibition of a trypsin-like activity from larvae of corn borer (Ostrinia nubilalis) using reverse micelles

Summary Endoproteinase(s) was isolated from a freeze-dried powder of larvae of Ostrinia nubilalis using reverse micellar solutions. The inhibition of proteinase was studied in reverse micelles with commercial Bowman-Birk soybean trypsin inhibitor and three trypsin inhibitors recently isolated from ripe cruciferous seeds.     

Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae)

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.     

The influence of planting date,transgenic Bt maize and hybrid relative maturity on European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) ovipositional patterns

A three year study was carried out at Hoytville and at Wooster, Ohio, USA from 2006 to 2008 to investigate the influence of planting date, transgenic maize and hybrid maturity on Ostrinia nubilalis (Hubner) population dynamics and oviposition patterns. Maize plants were planted in late April or early May, mid‐May and early June during each year. The moth flight pattern showed bivoltine generations during the three years. The first moth flight peaked in June, with the populations declining during July. The second moth flight peaked in August and declined towards the end of September or early October. Egg mass density did not differ significantly between transgenic and non‐transgenic maize of different maturities. Significant differences were observed, however, among planting dates, sampling dates, and sampling date × planting date interactions. Generally higher numbers of egg masses from second generation moths were deposited on late planted maize than middle and early plantings.     

Western corn rootworm and Bt maize: challenges of pest resistance in the field

Crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) manage many key insect pests while reducing the use of conventional insecticides. One of the primary pests targeted by Bt maize in the United States is the western corn rootworm, Diabrotica virgifera virgifera LeConte. Beginning in 2009, populations of western corn rootworm were identified in Iowa, USA that imposed severe root injury to Cry3Bb1 maize. Subsequent laboratory bioassays revealed that these populations were resistant to Cry3Bb1 maize, with survival on Cry3Bb1 maize that was three times higher than populations not associated with such injury. Here we report the results of research that began in 2010 when western corn rootworm were sampled from 14 fields in Iowa, half of which had root injury to Cry3Bb1 maize of greater than 1 node. Of these samples, sufficient eggs were collected to conduct bioassays on seven populations. Laboratory bioassays revealed that these 2010 populations had survival on Cry3Bb1 maize that was 11 times higher and significantly greater than that of control populations, which were brought into the laboratory prior to the commercialization of Bt maize for control of corn rootworm. Additionally, the developmental delays observed for control populations on Cry3Bb1 maize were greatly diminished for 2010 populations. All 2010 populations evaluated in bioassays came from fields with a history of continuous maize production and between 3 and 7 y of Cry3Bb1 maize cultivation. Resistance to Cry34/35Ab1 maize was not detected and there was no correlation between survival on Cry3Bb1 maize and Cry34/35Ab1 maize, suggesting a lack of cross resistance between these Bt toxins. Effectively dealing with the challenge of field-evolved resistance to Bt maize by western corn rootworm will require better adherence to the principles of integrated pest management.     

Molecular differentiation at nuclear loci in French host races of the European corn borer (Ostrinia nubilalis)

French populations of the European corn borer consist of two sympatric and genetically differentiated host races. As such, they are well suited to study processes that could be involved in sympatric speciation, but the initial conditions of host-race divergence need to be elucidated. Gene genealogies can provide insight into the processes involved in speciation. We used DNA sequences of four nuclear genes to (1) document the genetic structure of the two French host races previously delineated with allozyme markers, (2) find genes directly or indirectly involved in reproductive isolation between host races, and (3) estimate the time since divergence of the two taxa and see whether this estimate is compatible with this divergence being the result of a host shift onto maize after its introduction into Europe approximately 500 years ago. Gene genealogies revealed extensive shared polymorphism, but confirmed the previously observed genetic differentiation between the two host races. Significant departures from the predictions of neutral molecular evolution models were detected at three loci but were apparently unrelated to reproductive isolation between host races. Estimates of time since divergence between French host races varied from approximately 75,000 to approximately 150,000 years, suggesting that the two taxa diverged recently but probably long before the introduction of maize into Europe.     

QTL mapping for resistance against the European corn borer (Ostrinia nubilalis H.) in early maturing European dent germplasm

The European corn borer (ECB, Ostrinia nubilalis Hübner) is a major pest of maize in Central Europe. We mapped and characterized quantitative trait loci (QTLs) involved in resistance of maize against ECB damage, compared them with QTLs for agronomic traits, and evaluated the usefulness of marker-assisted selection (MAS) for improving ECB resistance in early maturing European maize germplasm. A total 226 F₃ families from the cross D06 (resistant) × D408 (susceptible), together with 93 RFLP and two SSR markers were used for the QTL analyses. For each F₃ family we measured the length of tunnels produced by larval stalk mining (TL), stalk damage ratings (SDR), and relative grain yield (RGY) in field experiments, with two replications in two environments in 1 year. The agronomic traits comprised grain yield under insecticide protection (GYP) and manual ECB larval infestation (GYI), the date of anthesis (ANT), and the in vitro digestibility of organic matter (IVDOM) of stover. Estimates of genotypic variance (σ² _g) were highly significant for all traits. Six QTLs for TL and five QTLs for SDR were detected, explaining about 50.0% of σ² _g. Most QTLs showed additive gene action for TL and dominance for SDR. No QTL was found for RGY. The number of QTLs detected for the agronomic traits ranged from two for GYI to 12 for ANT, explaining 12.5 to 57.3% of σ² _g, respectively. Only a single QTL was in common between the two resistance traits, as expected from the moderate trait correlation and the moderate proportions of σ² _g explained. Based on these results, MAS for improving ECB resistance can be competitive when cost-effective PCR-based marker systems are applied. However, it remains to be established whether the putative QTL regions for ECB resistance detected in the population D06 × D408 are consistent across other early maturing European maize germplasms. Received: 20 December 1999 / Accepted: 6 June 2000     

Age- and density-related oviposition behavior of the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Age-related oviposition patterns ofOstrinia nubilalis were studied at three population densities in the laboratory by releasing newly eclosed adults in wire-screened cages and analyzing their oviposition throughout the adult stage with digital analysis. Oviposition sequences of individual females depositing egg masses were documented on the third and seventh nights after eclosion with a video camcorder. During a sequence, a female produced an egg in an average time of 15 or 26 s on the third and seventh nights, respectively, and completed depositing an egg mass the size of 20–39 eggs in an average time of 316 and 525 s, respectively. Females were not easily disturbed during egg mass deposition and pulsated their abdomen before deposition of each egg. Females produced few egg masses the first night after eclosion. Oviposition increased on nights 2 and 3 but declined steadily thereafter as females matured. Females older than 6 nights produced fewer egg masses; the proportion of egg masses with fewer than 20 eggs increased gradually. By the end of the adults' lifetime, nearly 100% of the egg masses had fewer than 20 eggs. The data are fundamental to our research to define the role of phytochemicals in modifying oviposition behavior of the European corn borer.     

Resistance monitoring of field populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain

Approximately 22 000 hectares (5% of the total maize growing area) of transgenic maize expressing the Cry1Ab toxin from Bacillus thuringiensis (Bt maize) have been planted annually in Spain since 1998. Changes in the susceptibility to Cry1Ab of Spanish populations of the Mediterranean corn borer (MCB), Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), and the European corn borer (ECB), Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), were assessed by annual monitoring on Bt maize fields. No increase in resistance was detected in the MCB populations from Ebro, Albacete, and Badajoz, nor in the ECB populations from Ebro and Badajoz during the period 1999–2002. The susceptibility of the MCB population from Madrid fluctuated from year to year, but a gradual trend towards higher levels of tolerance was not observed. Laboratory selection assays for eight generations yielded selected strains of MCB and ECB that were 21‐ and 10‐fold significantly more tolerant to Cry1Ab than the corresponding unselected strains, respectively. Nevertheless, none of the field‐collected or laboratory‐selected larvae were able to survive on Bt maize. Considering these data, no consistent shifts in susceptibility were found for Spanish populations of MCB nor ECB after 5 years of Bt maize cultivation, but systematic field monitoring needs to be continued.     

Insights into genome differentiation: pheromone-binding protein variation and population history in the European corn borer (Ostrinia nubilalis)

Examination of sequence variation at nuclear loci can give insights into population history and gene flow that cannot be derived from other commonly used molecular markers, such as allozymes. Here, we report on sequence variation at a single nuclear locus, the pheromone-binding protein (PBP) locus, in the European corn borer (Ostrinia nubilalis). The European corn borer has been divided into three races in New York State on the basis of differences in pheromone communication and life history. Previous allozyme data have suggested that there is a small but significant amount of genetic differentiation between these races. The PBP does not appear to be involved in the pheromone differences between these races. Examination of variation at the PBP locus in the three races reveals no fixed differences between races despite high levels of polymorphism. There also appears to have been considerable recombination in the history of the pheromone-binding protein alleles. Observation of both recombination between alleles and lack of significant nucleotide or insertion/deletion divergence between races leads us to suggest that these populations are either recently diverged or have continued to exchange genetic material subsequent to divergence in pheromone communication and life history.     

Genetic mapping of sexual isolation between E and Z pheromone strains of the european corn Borer (Ostrinia nubilalis)

The E and Z pheromone strains of the European corn borer (ECB) provide an exceptional model system for examining the genetic basis of sexual isolation. Differences at two major genes account for variation in female pheromone production and male behavioral response, components of the pheromone communication system known to be important for mate recognition and mate choice. Strains of ECB are morphologically indistinguishable, and surveys of allozyme and DNA sequence variation have revealed significant allele frequency differences at only a single sex-linked locus, Tpi. Here we present a detailed genetic linkage map of ECB using AFLP and microsatellite markers and map the factors responsible for pheromone production (Pher) and male response (Resp). Our map covers 1697 cM and identifies all 31 linkage groups in ECB. Both Resp and Tpi map to the Z (sex) chromosome, but the distance between these markers (>20 cM) argues against the hypothesis that patterns of variation at Tpi are explained by tight linkage to this "speciation gene." However, we show, through analysis of marker density, that Tpi is located in a region of low recombination and suggest that a second Z-linked reproductive barrier could be responsible for the origin and/or persistence of differentiation at Tpi.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Effectiveness of Trichogramma ostriniae and T. nubilale for controlling the European corn borer Ostrinia nubilalis in sweet corn

Seven field releases of Trichogramma ostriniae and T. nubilale (Hymenoptera: Trichogrammatidae) were made separately and in combination in a sweet corn field to compare the level of parasitism in sentinel eggs of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). The results indicate that the levels of egg parasitism among different release dates differed mainly because of changes in weather and plant architecture within the season. The level of egg parasitism by releasing T. ostriniae alone was found to be 15% higher than that by releasing T. nubilale alone, and 20% higher than by releasing the combination of the two species. Further analyses using the logistic regression model for independent and correlated data indicated T. ostriniae to be more efficient at discovering host egg masses and to have higher levels of egg parasitism than T. nubilale. Mutual interference between T. ostriniae and T. nubilale was the main factor for the lower level of egg parasitism when T. ostriniae and T. nubilale were released together. The results suggest that T. ostriniae is the better candidate for augmentative releases for control of the European corn borer, and the two species should not be released into a corn field at the same time.     

Effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in European corn borer (Ostrinia nubilalis) larvae

European corn borer (ECB; Ostrinia nubilalis (Hubner)) larvae (third instar) fed 0.05% w/w wheat germ agglutinin (WGA) in their diet for 72 h showed very little increase in body weight, whereas weight of control larvae increased nearly fourfold. Light and transmission electron microscopy studies showed that the morphology of the peritrophic membrane (PM) changed within 24 h after ECB larvae fed on the WGA diet. Whereas the PM in the anterior region of the midgut was a thin membranous structure in control larvae, the WGA-fed larvae secreted a multiple-layered and unorganized PM that contained embedded food particles, bacteria, and pieces of disintegrated microvilli. Gold-labeled WGA was localized specifically in the PM and microvilli. The PM of WGA-fed larvae was inundated with dark-staining amorphous structures that, when incubated with anti-WGA, showed heavy WGA localization. The antibody label indicated that most of the ingested WGA was found in the PM, with lesser amounts on the microvillar surface and the least amount within the epithelium. After 72 h, the middle portion of the mesenteron revealed a thin, compact PM in the control larvae, whereas the PM of the WGA-fed larvae was multilayered and discontinuous, which allowed plant cell-wall fragments to penetrate into the microvilli of the epithelium. Scanning electron microscopy of PMs from fifth instar ECB larvae fed the WGA diet revealed a breakdown in the chitinous meshwork by 48 h after initiation of feeding. The endo-PM surface from control larvae was smooth and intact, whereas the PM of WGA-fed larvae showed disintegration of the meshwork and a reduced proteinaceous matrix. This allowed bacteria and food particles to penetrate through the PM into the ectoperitrophic space and directly contact the microvilli. Therefore, WGA, a protein inhibitor of larval growth, interferes with the formation and integrity of the PM, which exposes the brush border to ingested material. This, in turn, appears to stimulate secretion of additional PM layers, the concomitant disintegration of the microvilli, and cessation of feeding.