Identification and expression profile of odorant-binding proteins in Halyomorpha halys (Hemiptera: Pentatomidae).

The brown marmorated stink bug, Halyomorpha halys, is a devastating invasive species in the USA. Similar to other insects, olfaction plays an important role in its survival and reproduction. As odorant-binding proteins (OBPs) are involved in the initial semiochemical recognition steps, we used RNA-Sequencing (RNA-Seq) to identify OBPs in its antennae, and studied their expression pattern in different body parts under semiochemical stimulation by either aggregation or alarm pheromone or food odorants. Thirty full-length putative HhalOBPs were identified, corresponding to 22 'classic' OBPs and eight 'Plus-C' OBPs. The similarity amongst them ranged from 4.95-70.92%, and with another 325 hemipteran OBPs similarity ranged from 1.94-91.51%, the highest levels being with other stink bug OBPs. Phylogenetic analysis confirmed the monophyly of seven groups of stink bug and other hemipteran OBPs. All 30 HhalOBPs were expressed and about 2/3 were expressed primarily in antennae. The expression of 21 HhalOBPs was higher in the antennae under alarm pheromone stimulus, indicating that multiple OBPs may be responding to this pheromone. Two were highest in antennae under aggregation pheromone stimulus. These findings should provide a basis for understanding the physiological functions of HhalOBPs and the chemosensory perception of this pest, which may help to uncover new control targets for behavioural interference.

Planting patterns of in-field refuges observed for Bt maize in Minnesota.

The U.S. Environmental Protection Agency (USEPA) requires the use of nontransgenic refuges to slow the evolution of insect resistance to transgenic crops. In-field refuges, or refuges that are planted within the same field as the transgenic crop, are allowed; however, these refuges are required to be at least four rows wide. We described in-field planting patterns used by growers for both CrylAb [against Ostrinia nubilalis (Hübner)] and Cry3Bb (against Diabrotica virgifera virgifera LeConte) maize, Zea mays L. Maize fields known to contain Cry1Ab, Cry3Bb, or both were sampled in southwestern Minnesota during late June and early September 2005. Rows were sampled to describe the pattern of in-field refuges in the entire field. Most in-field refuges contained > 20% Cry seed (79% of Cry1Ab and 84% of Cry3Bb). However, only 5% of Cry1Ab fields and 2% of Cry3Bb fields with in-field refuges were in compliance with USEPA requirements because the Cry- seed was not in wide enough strips or blocks. Most growers had planted their fields with either finely mixed refuges or with strips that were too narrow. There was a high diversity in planting patterns, and the occurrence of Cry seed was in random rows. Growers may have been rushed while planting and not noticed which seed was going into which rows. Resistance failures have not been documented for either O. nubilalis or D. virgifera virgifera, so better education programs will need to be undertaken to encourage growers to plant in-field refuges properly.

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).

Managing the evolution of insect resistance to transgenic plants.

The evolution of resistance in pests such as the European corn borer will imperil transgenic maize varieties that express insecticidal crystal proteins of Bacillus thuringiensis. Patchworks of treated and untreated fields can delay the evolution of pesticide resistance, but the untreated refuge fields are likely to sustain heavy damage. A strategy that exploits corn borer preferences and movements can eliminate this problem. Computer simulation indicates that this approach can delay the evolution of resistance and reduce insect damage in the untreated fields of a patchwork planting regime.

Microbial populations and enzyme activities in soil in situ under transgenic corn expressing cry proteins from Bacillus thuringiensis.

Transgenic Bt crops produce insecticidal Cry proteins that are released to soil in plant residues, root exudates, and pollen and that may affect soil microorganisms. As a continuation of studies in the laboratory and a plant-growth room, a field study was conducted at the Rosemount Experiment Station of the University of Minnesota. Three Bt corn varieties that express the Cry1Ab protein, which is toxic to the European corn borer (Ostrinia nubilalis Hübner), and one Bt corn variety that expresses the Cry3Bb1 protein, which is toxic to the corn rootworm complex (Diabrotica spp.), and their near-isogenic non-Bt varieties were evaluated for their effects on microbial diversity by classical dilution plating and molecular (polymerase chain reaction-denaturing gradient gel electrophoresis) techniques and for the activities of some enzymes (arylsulfatases, acid and alkaline phosphatases, dehydrogenases, and proteases) involved in the degradation of plant biomass. After 4 consecutive years of corn cultivation (2003-2006), there were, in general, no consistent statistically significant differences in the numbers of different groups of microorganisms, the activities of the enzymes, and the pH between soils planted with Bt and non-Bt corn. Numbers and types of microorganisms and enzyme activities differed with season and with the varieties of corn, but these differences were not related to the presence of the Cry proteins in soil. The Cry1Ab protein of Bt corn (events Bt11 and MON810) was detected in most soils during the 4 yr, whereas the Cry3Bb1 protein was not detected in soils of Bt corn (event MON863) expressing the cry3Bb1 gene.

Frequency of resistance to Bacillus thuringiensis toxin CrylAb in Greek and Spanish population of Sesamia nonagrioides (Lepidoptera: Noctuidae).

The high-dose/refuge strategy is considered as the main strategy for delaying resistance in target pests to genetically modified crops that produce insecticidal proteins derived from Bacillus thuringiensis Berliner. This strategy is based on a key assumption that resistance alleles are initially rare (<10(-3)). To test this assumption, we used an F2 screen on natural populations of Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) from Greece and Spain. In total, 75 lines from Greece and 85 lines from Spain were screened for survival of F2 larvae on Cry1Ab corn, Zea mays L., leaves. No major resistance alleles were found. The frequency of resistance alleles in the Greek population was <9.7 x 10(-3) with 95% probability, which was very similar to that of the Spanish population (<8.6 x 10(-3) with 95% probability), and the expected frequencies were 3.2 x 10(-3) (0-0.0097) and 2.9 x 10(-3) (0-0.0086) in Greece and Spain (pooled 1.5 x 10(-3)). The experiment-wise detection probability of resistance was 94.0 and 97.5% for the Greek and the Spanish population, respectively. Evidence of alleles conferring partial resistance to Cry1Ab was found only for the Greek population. The frequency of alleles for partial resistance was estimated as 6.5 x 10(-3) with a 95% credibility interval between 8 x 10(-4) and 17.8 x 10(-3) and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to CrylAb, regarding the population of S. nonagrioides, may be rare enough so that the high-dose/refuge strategy could be applied with success for resistance management.

Assessing environmental risks of transgenic plants.

By the end of the 1980s, a broad consensus had developed that there were potential environmental risks of transgenic plants requiring assessment and that this assessment must be done on a case-by-case basis, taking into account the transgene, recipient organism, intended environment of release, and the frequency and scale of the intended introduction. Since 1990, there have been gradual but substantial changes in the environmental risk assessment process. In this review, we focus on changes in the assessment of risks associated with non-target species and biodiversity, gene flow, and the evolution of resistance. Non-target risk assessment now focuses on risks of transgenic plants to the intended local environment of release. Measurements of gene flow indicate that it occurs at higher rates than believed in the early 1990s, mathematical theory is beginning to clarify expectations of risks associated with gene flow, and management methods are being developed to reduce gene flow and possibly mitigate its effects. Insect pest resistance risks are now managed using a high-dose/refuge or a refuge-only strategy, and the present research focuses on monitoring for resistance and encouraging compliance to requirements. We synthesize previous models for tiering risk assessment and propose a general model for tiering. Future transgenic crops are likely to pose greater challenges for risk assessment, and meeting these challenges will be crucial in developing a scientifically coherent risk assessment framework. Scientific understanding of the factors affecting environmental risk is still nascent, and environmental scientists need to help improve environmental risk assessment.

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern United States Corn Belt population of European corn borer (Lepidoptera: Crambidae).

The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F2 screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10(-3)) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F2 screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.

Lady Beetle (Coleoptera: Coccinellidae) Communities in Soybean and Maize.

Coccinellids provide the most effective natural control of soybean aphid, but outbreaks remain common. Previous work suggests that native coccinellids are rare in soybean, potentially limiting soybean aphid control. We compared the coccinellid community in soybean with that of maize to identify differences in how coccinellid species use these habitats. As maize has long been used by coccinellids in the Americas, we hypothesized that coccinellids native to the Americas would use maize habitats, while exotic coccinellids would be more common in soybean. We identified and quantified aphids and all species and stages of coccinellids in a randomized complete block experiment with four blocks of 10 by 10 -m plots of soybean and maize in central Minnesota during 2008 and 2009. Coccinellid egg masses were identified by hatching in the laboratory. We used repeated-measures ANOVA to identify the dominant species in each habitat and compared species richness and Shannon's diversity with a paired t-test. Aphids and coccinellids had a similar phenology across habitats, but the coccinellid species composition differed significantly between soybean and maize. In soybean, the exotic, Harmonia axyridis Pallas, was the dominant species, while in maize, H. axyridis and the native, Coleomegilla maculata De Geer, were co-dominant. Eggs of H. axyridis were abundant in both habitats. In contrast, C. maculata eggs were very rare in soybean, despite being abundant in adjacent plots of maize. Species diversity was higher in maize. These findings were consistent with other published studies of coccinellid communities in these habitats.

Plant structural complexity and host-finding by a parasitoid.

There are three major components to plant structure relevant to searching parasitoids: 1) plant size or surface area, 2) the variation among plant parts (structural heterogeneity), such as seed heads, flowers and nectaries, and heterogeneous surfaces (e.g. glabrous, hirsute), and 3) the connectivity of parts or plant form (structural complexity). We examined the effect of structural complexity, while controlling for size and structural heterogeneity, on searching behaviors of Trichogramma nubilale in controlled environments. Females were presented with a structurally simple surface and a structurally complex one. Parasitism rates were 2.9 times higher on simple surfaces than on complex ones. Unexpectedly, when no hosts were present, searching time on simple surfaces was 1.2 times higher than on complex surfaces. This implies that structural complexity per se can affect the giving-up-time of a searching parasitoid. Searching efficiency, however, was the dominant process, and females found hosts on simple surfaces 2.4 times faster than on complex surfaces. Structural complexity can have a dramatic effect on the success of parasitoid search.

F2 screen variations and associated statistics.

The F2 screen is a flexible methodology used to estimate the frequency of resistance alleles (R) in an insect population. We have developed several alternative protocols, along with the associated statistics, to conduct an F2 screen with mated or unmated individuals, random and nonrandom mating of F1 adults, and the screening of multiple lines together in the F2 screen. Our protocols describe how to perform and analyze an F2 screen starting with unmated P1 as an alternative to mated females. A randomly mated population of > or = 50 F1 adults should be sufficient to detect R alleles > 99% of the time. If nonrandom mating occurs in the F2 screen, it is most likely to be positive assortative mating, and this would improve the probability of detecting an R allele. Pair mating the F1 adults greatly increases costs of the screen while providing a small, but negligible improvement in detecting R alleles. The number of screens may be reduced by more than two-thirds by screening multiple lines together. These methodological variants show the F2 screen to be much more robust than originally described.

An in-field screen for early detection and monitoring of insect resistance to Bacillus thuringiensis in transgenic crops.

We present a field-based approach to detect and monitor insects with resistance to insecticidal toxins produced by transgenic plants. Our objective is to estimate the phenotypic frequency of resistance in a population by relating the densities of insects on genetically transformed plants to densities on nontransformed plants. We focus on European corn borer, Ostrinia nubilalis (Hübner), in sweet corn, Zea mays L., expressing Cry1Ab from Bacillus thuringiensis subsp. kurstaki Berliner to illustrate principles underlying the method. The probability of detecting one or more rare, resistant larvae depends on sample size, the density of larvae on nontransformed plants, and an assumed frequency of resistant phenotypes in a given population. Probability of detection increases with increases in sample size, background density, or the frequency of resistant individuals. Following binomial probability theory, if a frequency of 10(-4) is expected, 10(3)-10(4) samples must be collected from a B. thuringiensis (Bt) crop to have at least a 95% probability of locating one or more resistant larvae. In-field screens using transgenic crops have several advantages over traditional laboratory-based methods, including exposure to a large number of feral insects, discrimination of resistant individuals based on Bt dosages expressed in the field, incorporation of natural and Bt-induced mortality factors, simultaneous monitoring for more than one insect species, and ease of use. The approach is amenable to field survey crews working in research, extension, and within the seed corn industry. Estimates of the phenotypic frequency of resistance from the in-field screen can be useful for estimating initial frequency of resistant alleles. Bayesian statistical methods are outlined to estimate phenotype frequencies, allele frequencies, and associated confidence intervals from field data. Results of the approach are discussed relative to existing complementary methods currently available for O. nubilalis and corn earworm, Helicoverpa zea (Boddie).

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae).

The refuge plus high-dose strategy for resistance management assumes that the frequency of resistance alleles is low. We used an F2 screen to estimate the frequency of resistance to transgenic corn that produces Bacillus thuringiensis Berliner Cry1Ab toxin (Bt corn) in an Iowa population of European corn borer, Ostrinia nubilalis (Hübner). We also proposed a modification to the statistical analysis of the F2 screen that extends its application for nonuniform prior distributions and for repeated sampling of a single population. Based on a sample of 188 isofemale lines derived from females caught at light traps during the 2nd flight of 1997, we show with 95% confidence that the frequency of resistance to Bt corn was <3.9 x 10(-3) in this Iowa population. These results provide weak evidence that the refuge plus high-dose strategy may be effective for managing resistance in O. nubilalis to Bt corn. Partial resistance to Cry1Ab toxin was found commonly. The 95% CI for the frequency of partial resistance were [8.2 x 10(-4), 9.4 x 10(-3)] for the Iowa population. Variable costs of the method were 14.90 dollars per isofemale line, which was a reduction of 25% compared with our initial estimate.

Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae).

Using the F2 screen methodology, we estimated the frequency of alleles conferring resistance to the Cry1Ab toxin of Bacillus thuringiensis Berliner in a Philippine population of the stem borer Scirpophaga incertulas (Walker). Evaluation of >450 isofemale lines for survival of F2 larvae on cry1Ab plants did not detect the presence of an allele conferring a high level of resistance. The frequency of such an allele in the sampled population was conservatively estimated to be <3.6 x 10(-3) with 95% confidence and a detection probability of 94%. However, there was evidence of the presence of alleles conferring partial resistance to Cry1Ab. The frequency of alleles for partial resistance was estimated as 4.8 x 10(-3) with a 95% CI between 1.3 x 10(-3) and 1.04 x 10(-2) and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab in the population of S. incertulas sampled is not too high to preclude successful implementation of the high dose/refuge resistance management strategy.

Benefits of self-superparasitism in a polyembryonic parasitoid.

Macrocentrus grandii is a polyembryonic parasitoid, with embryos that divide asexually within the host (European corn borer, Ostrinia nubilalis) to produce broods of clonal offspring. From a biological control standpoint, polyembryony seems advantageous because each parasitized host yields multiple parasitoids with minimal time and egg investment. When we observed M. grandii in the field, however, we found that the parasitoid virtually always invested additional time and, if possible, stings into hosts that it had already stung, apparently reducing some of the advantages of polyembryony. We therefore investigated and found support for two potential benefits that can be gained by self-superparasitism in this system. First, multiple stings allowed production of mixed-sex broods: 27% of multiply-stung versus 0% of singly-stung hosts produced mixed-sex broods. Second, multiple stings increased mean parasitoid progeny produced per host, primarily by reducing the chance of complete brood failure. Our results indicate substantial benefit for a second sting, but little benefit for three or more stings, even though M. grandii was sometimes observed to invest more than two stings within a single host. However, we also found that within-host larval competition is prevalent, suggesting that supernumerary stings may pay off in competition against conspecific larvae. Such additional investment within a single host would be particularly beneficial when hosts, rather than eggs, are limiting, but would decrease the overall efficacy of M. grandii as a biological control agent.

Foraging for intermittently refuged prey: theory and field observations of a parasitoid.

Many insect herbivores feed in concealed locations but become accessible intermittently, creating windows of greater vulnerability to attack, and generating a proportion of the prey population that is readily accessible to foraging natural enemies. We incorporated accessible prey into an extant optimal foraging model, and found that this addition allowed opportunistic exploitation of prey that have already emerged from refugia (the leaving strategy) as a viable strategy, in addition to waiting at refugia for prey to emerge (the waiting strategy). We parameterized the model empirically for the parasitoid Macrocentrus grandii and its host, Ostrinia nubilalis, under field conditions. The model predicted that M. grandii should adopt a leaving strategy when host patch density is high (travel time between patches is short), but a waiting strategy when host patch density is low (travel time between patches is long). Field observations of M. grandii patch tenure were consistent with model predictions, indicating that M. grandii exhibited flexible behaviour based on experience within a foraging bout, and that these behavioural shifts improved foraging efficiency. Behaviour of M. grandii was responsive to heterogeneity in host emergence rates, and appeared to be driven by the relatively small proportion of the host population that became accessible at a fast rate. Therefore understanding forager responses to intermittently refuged prey may require characterization of the behaviour of a subset of the prey population, rather than the average prey individual. The model can potentially be used as a framework for comparative studies across forager taxa, to understand when foragers on intermittently accessible prey should adopt fixed waiting or leaving strategies vs. a flexible strategy that is responsive to the current environment.

F2 screen for resistance to a Bacillus thuringiensis-maize hybrid in the sugarcane borer (Lepidoptera: Crambidae).

A novel F2 screening technique was developed for detecting resistance in sugarcane borer, Diatraea saccharalis (F.), to transgenic Bacillus thuringiensis (Bt)-maize expressing the Cry1Ab insecticidal protein. The F2 screening method involved (i) collecting larvae from maize fields; (ii) establishing two-parent families; (iii) screening F2 neonates for survival on Bt-maize leaf tissues; and (iv) confirming resistance on commercial Bt-maize plants. With the F2 screening method, 213 iso-line families of D. saccharalis were established from field collections in northeast Louisiana, USA and were screened for Bt resistance. One family was confirmed to carry a major Bt resistance allele(s). In a laboratory bioassay, larval mortality of the Bt-resistant D. saccharalis on Bt-maize leaf tissues was significantly lower than that of a Bt-susceptible strain. This Bt-resistant D. saccharalis population is the first corn stalk borer species that has completed larval development on commercial Bt-maize. The F2 screening protocol developed in this study could be modified for detecting Bt resistance alleles in other similar corn stalk borers, such as the European corn borer, Ostrinia nubilalis (Hübner), and the southwestern corn borer, D. grandiosella Dyar.

Habitat modification contributes to associational resistance between herbivores.

Associational resistance, in which one species gains protection from its consumers by association with a competitor, is common among plants but has seldom been documented among insects. Here we show that parasitism of an aboveground herbivore, European corn borer, by its specialist parasitoid Macrocentrus grandii, is reduced 98% in the presence of a belowground herbivore, corn rootworm. We tested the hypothesis that this positive indirect interaction between corn rootworm and corn borer was mediated by corn rootworm's influence on the habitat: plant height was diminished by 33% and plant density by 20%, resulting in a more open habitat. We found that M. grandii showed a two- to five-fold preference for dense versus open habitats, and that experimental reduction of plant density in the absence of corn rootworm reduced M. grandii parasitism of corn borer by 13%. This result supports the presence of a habitat modification effect as a contributing factor to associational resistance for corn borer. We argue that associational resistance may be more common among phytophagous insects than previously appreciated.