Invasion of pests resistant to Bt toxins can lead to inherent non-uniqueness in genetically modified Bt-plant dynamics: mathematical modeling

Genetically modified crops are effective pest management tools for worldwide growers. However, there is a concern that pests may develop resistance to Bt-toxins produced by genetically modified Bt-plants. We study the impact of the Bt-resistant pests on Bt-crops. Furthermore, the dynamics of the Bt-plant-Bt-susceptible insects-Bt-resistant insects system is analysed and it is shown that throughout the insect reproduction period the plant biomass dynamics resulting from invasion of Bt-resistant insects is non-unique. Namely, the chaotic attractor and the limit cycle, which are responsible for the plant and insect biomass dynamics, are shown to coexist. As a result, the Bt-plant-Bt-resistant insect system can manifest either chaotic or regular oscillations of plant and insect biomass depending on spatial patterns resulting from invasion of Bt resistant insects into the Bt plant-Bt susceptible insect system. We show that the non-uniqueness of the system dynamics under unfavorable environmental conditions, such as in the so-called zones of risky agriculture in many developing countries and industrialized countries, can lead to essential decrease in the plant biomass.     

Beyond Bt resistance of pests in the context of population dynamical complexity

Complexity in ecological systems often prevents long-term predictions about changes in population size and properties of the population dynamics. Mathematical modeling of such complex system behaviors can provide a rough idea of scenarios of the population dynamics. We use the reaction–diffusion model [Medvinsky, A.B., Morozov, A.Y., Velkov, V.V., Li, B.-L., Sokolov, M.S., Malchow, H., 2004. Modeling the invasion of recessive Bt-resistant insects: an impact on transgenic plants. J. Theor. Biol. 231, 121–127] to study the impact of pests resistant to toxins produced by genetically modified plants on the dynamics of the plant–insect system. Using genetically modified crops is an effective pest management tool for world-wide growers. However, there is a concern that pests may develop resistance to Bt toxins, which are a product of Bacillus thuringiensis genes introduced into genetically modified Bt plants. We show by computer simulations that the Bt plant–Bt-resistant insect dynamics resulting from the invasion of the Bt-resistant pests leads to variety of complex changes in the plant–insect biomass, which underlie the dependence of the Bt plant biomass on the duration of the insect reproduction period. We demonstrate that the plant and insect biomass can undergo both regular and irregular oscillations. We show that the character of such oscillations essentially depends on local insect fluxes resulting from inhomogeneous spatial distributions of the insects. In order to characterize the insect diffusion fluxes we introduce a new parameter, the diffusion number Dn. We show that the dependence between a value of Dn and regularity/irregularity of the plant–insect biomass oscillations is governed by a region in the model parameter space. In one of the regions the growth of the value of the diffusion number correlates with the transformation of regular oscillations into irregular ones, while in the neighboring region of the model parameter space the dependence between the character of the plant–insect oscillations and the value of the diffusion number Dn is more complex. The oscillations are irregular if the values of Dn are between 0.05 and 0.25. On either side of this interval the plant–insect oscillations are regular. The complex character of the response of the Bt crop–pest system to the invasion of Bt-resistant insects can lead to significant complications in attempts to regulate the system dynamics.     

Effect of the high dose/refuge strategy on Bt-crop yield dynamics (mathematical simulation)

A mathematical model is presented that describes interactions between Bt-crops and insect pests taking into account the plant growth rate, consumption of plant biomass by pests, suppression of insect proliferation by Bt toxins, emergence of Bt-resistant insects and their mixing with the wild type. It is shown that migration of Bt-susceptible insects from “refuge plots” onto the Bt-crop field invaded by resistance-carrying insects improves the yield dynamics; being high enough, this inflow eventually eliminates the resistant pest population.     

Dynamics of Bt-crop biomass upon invasion of a Bt-resistant insect pest: A mathematical model

Simulation of some consequences of invasion of a Bt-resistant insect pest into an agricultural eco-system containing a Bt crop shows that such invasion alters the plant biomass dynamics, decreases the crop yield, and impairs yield predictability. The yield is poorly predictable in a small region of parameter values, which depends on the duration of the insect reproductive period relative to the crop-growing season.

     

The impact of the high dose-refuge strategy on the Bt-crop yield. Mathematical simulations

A mathematical model is presented, which describes interactions between Bt-plants and pests in terms of the plant growth rate, plant annual reseeding, consumption of plant biomass by pests, the action of Bt toxins, and the emergence of Bt-resistant insects. It has been shown that the migration of Bt-susceptible insects affects the Bt-crop yield dynamics and provides the exclusion of Bt-resistant pests if the migration exceeds the threshold value.     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

Insect resistance to Bacillus thuringiensis: alterations in the indianmeal moth larval gut proteome

Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.     

Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests. Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control. This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments. C. plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide. Bt oilseed rape caused 100% mortality of a Bt-susceptible P. xylostella strain but no mortality of the Bt-resistant P. xylostella strain NO-QA. C. plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C. plutellae larvae to complete their development. In contrast, C. plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons. Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments. The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.     

Fitness costs of resistance to Bacillus thuringiensis in the Indianmeal moth, Plodia interpunctella

Genetic changes in insects that result in insecticide resistance can also affect their fitness. Here, we report measurements of development time and survival of the Indianmeal moth, Plodia interpunctella (Hübner), to compare the relative fitness of Bacillus thuringiensis (Bt)-susceptible and -resistant colonies. Measurements of larval development time and survival indicated that a fitness cost was associated with resistance to Bt in some Bt-resistant colonies but not others. Comparisons of geographically different populations revealed inherent differences in development time and survival. In most cases, Bt-resistant moths suffered no disadvantage when feeding on a Bt-treated diet. In many cases, the development of Bt-resistant moths on Bt-treated diet was slower than the unselected moths on untreated diet, but it is unclear whether these differences would affect the successful mating of susceptible and resistant moths.     

Astylus atromaculatus (Coleoptera: Melyridae): abundance and role in pollen dispersal in Bt and non-Bt cotton in South Africa

In South Africa, modified Bt (Cry1 Ac) cotton cultivars and organic ones coexist. This raises the question of the risk of dissemination of genetically modified (GM) pollen to non-GM crops by visiting insects. We inventoried the flower-visiting insects in Bt and non-Bt cotton fields of the South African Highveld region and investigated their role in pollen dispersal. Their diversity and abundance varied slightly among sites, with Astylus atromaculatus as the predominant insect on both Bt and non-Bt cotton flowers. The other major flower-visiting species were Apis mellifera and solitary Apidae. No differences were found in the abundance of each taxum between Bt and non-Bt cotton except for Scoliidae and Nitidulidae, which were scarce overall (<0.5%) but more abundant on the non-Bt flowers in the central area of the field at one site. The pollen load on A. atromaculatus was as high as on Apis mellifera. Cage tests showed that A. atromaculatus can pollinate female cotton plants by transferring pollen from male donor plants. In the field, the flight range of this insect was generally short (25 m), but it can occasionally reach up to 200 m or even more. This study therefore highlights that A. atromaculatus, commonly regarded as a pest, could be an unexpected but efficient pollinator. Because its population density can be high, this species could mediate unwanted cotton pollen flow when distances between coexiting fields are not sufficient.     

Effect of Bt broccoli and resistant genotype of Plutella xylostella (Lepidoptera: Plutellidae) on life history and prey acceptance of the predator Coleomegilla maculata (Coleoptera: Coccinellidae)

The ecological implications on biological control of insecticidal transgenic plants, which produce crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), remains a contentious issue and affects risk assessment decisions. In this study, we used a unique system of resistant insects, Bt plants and a predator to critically evaluate this issue. The effects of broccoli type (normal or expressing Cry1Ac protein) and insect genotype (susceptible or Cry1Ac-resistant) of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined for their effects on the life history of the predator, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) over two generations. Additional behavioral studies were conducted on prey choice. C. maculata could not discriminate between Bt-resistant and susceptible genotypes of P. xylostella, nor between Bt and normal broccoli plants with resistant genotypes of P. xylostella feeding on them. The larval and pupal period, adult weight and fecundity of each female were not significantly different when C. maculata larvae fed on different genotypes (Bt-resistant or susceptible) of insect prey larvae reared on Bt or non-Bt broccoli plants. The life-history parameters of the subsequent generation of C. maculata fed on Bt broccoli-reared resistant P. xylostella were also not significantly different from those on non-Bt broccoli. These results indicated that Cry1Ac did not harm the life history or prey acceptance of an important predator after two generations of exposure. Plants expressing Cry1Ac are unlikely to affect this important predator in the field.     

How to cope with insect resistance to Bt toxins?

Transgenic Bt crops producing insecticidal crystalline proteins from Bacillus thuringiensis, so-called Cry toxins, have proved useful in controlling insect pests. However, the future of Bt crops is threatened by the evolution of insect resistance. Understanding how Bt toxins work and how insects become resistant will provide the basis for taking measures to counter resistance. Here we review possible mechanisms of resistance and different strategies to cope with resistance, such as expression of several toxins with different modes of action in the same plant, modified Cry toxins active against resistant insects, and the potential use of Cyt toxins or a fragment of cadherin receptor. These approaches should provide the means to assure the successful use of Bt crops for an extended period of time.     

A QTL that enhances and broadens Bt insect resistance in soybean

Effective strategies are needed to manage insect resistance to Bacillus thuringiensis (Bt) proteins expressed in transgenic crops. To evaluate a multiple resistance gene pyramiding strategy, eight soybean (Glycine max) lines possessing factorial combinations of two quantitative trait loci (QTLs) from plant introduction (PI) 229358 and a synthetic Bt cry1Ac gene were developed using marker-assisted selection with simple sequence repeat markers. Field studies were conducted in 2000 and 2001 to evaluate resistance to corn earworm (Helicoverpa zea) and soybean looper (Pseudoplusia includens), and detached leaf bioassays were used to test antibiosis resistance to Bt-resistant and Bt-susceptible strains of tobacco budworm (TBW; Heliothis virescens). Based on defoliation in the field and larval weight gain on detached leaves, lines carrying a combination of cry1Ac and the PI 229358 allele at a QTL on linkage group M were significantly more resistant to the lepidopteran pests, including the Bt-resistant TBW strain, than were the other lines. This is the first report of a complementary additive effect between a Bt transgene and a plant insect resistance QTL with an uncharacterized mode of action that was introgressed using marker-assisted selection.     

Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?

Bacillus thuringiensis endotoxins (Bt-toxins) are the most important biopesticides used in controlling insect pests and vectors of diseases. The emergence of widespread resistance to Bt in some insect species is a serious threat to agricultural production. Analysis of Bt-resistant and susceptible laboratory strains of Helicoverpa armigera revealed elevated immune responses involving increased melanization and the presence of a soluble toxin-binding glycoprotein in the hemolymph and gut lumen of the resistant strain. We propose a resistance mechanism against toxins based on a systemic immune-induction that can be transmitted to the next generation by a maternal effect.     

A meta-analysis of effects of Bt crops on honey bees (Hymenoptera: Apidae)

Background

Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations.

Methodology/Principal Findings

We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings.

Conclusions/Significance

Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator.     

Not all GMOs are crop plants: non-plant GMO applications in agriculture

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.     

Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure

A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.     

Effect of entomopathogenic nematodes on the fitness cost of resistance to Bt toxin crylac in pink bollworm (Lepidoptera: Gelechiidae)

The widespread use of crop plants genetically engineered to produce toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on insect populations to evolve resistance. The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major pest of cotton in the southwestern United States that is currently controlled with transgenic cotton that produces Bt toxin Cry1Ac. Previously reported theoretical work suggests that, in conjunction with a high dose/refuge strategy, fitness costs of Bt resistance can slow or prevent the evolution of resistance. We report here that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) increased the fitness cost of resistance to Cry1Ac in P. gossypiella. Mortality of P. gossypiella from fourth instar to adult eclosion was significantly higher for a Bt-resistant strain than a susceptible strain in tests with two to 14 infective juveniles of S. riobrave per larva, but it did not differ between strains when nematodes were absent. Nematodes established in P. gossypiella larvae at all concentrations tested, and nematode reproduction in infected P. gossypiella larvae occurred at nematode concentrations of four to 14 infective juveniles per larva. Our results suggest that incorporation of entomopathogenic nematodes into an integrated resistance management strategy could help to delay pest resistance to Bt toxins.     

转Bt基因抗虫作物对非靶标害虫的影响

转基因作物的长期大面积种植, 在为农业生产带来惠益的同时, 对农业生态系统的健康和稳定可能会产生潜在的影响。转基因作物表达的Bt蛋白对靶标害虫起到较好的控制效果, 而对Bt蛋白不敏感的非靶标害虫种群可能会迅速发展起来, 对作物造成为害。随着抗虫转基因作物的连续多年种植, 科学家们对于田间杀虫剂施用量的增减看法不尽一致。通过总结已有的研究报道, 本文以Bt玉米和Bt棉花为例, 分析了大田中非靶标害虫暴发的现状, 以及暴发的主要原因(如杀虫剂的使用、害虫天敌减少和物种替代)。在生产实践中, 抗虫作物的长期大面积释放导致广谱杀虫剂施用量减少, 田间非靶标害虫数量上升。因此今后需要继续开展更多的研究来综合评估种植转Bt基因作物产生的长期潜在影响, 优化害虫防治措施, 避免非靶标害虫暴发。     

Characterization of cDNAs encoding three trypsin-like proteinases and mRNA quantitative analysis in Bt-resistant and -susceptible strains of Ostrinia nubilalis

Our previous studies suggested that Bacillus thuringiensis (Bt) resistance in a Dipel-resistant strain of Ostrinia nubilalis was primarily due to reduced trypsin-like proteinase activity. In this study, we demonstrated a 254-fold resistance to Cry1Ab protoxin but only 12-fold to trypsin-activated Cry1Ab toxin in the Dipel-resistant strain. Significantly higher resistance to Cry1Ab protoxin than to trypsin-activated Cry1Ab toxin further supports the hypothesis that reduced trypsin-like proteinase activity leading to reduced activation of the Bt protoxin is a major resistance mechanism in the Dipel-resistant strain. To understand the molecular basis of reduced proteinase activity, three cDNAs, OnT2, OnT23, and OnT25, encoding full-length trypsin-like proteinases, were sequenced in Bt-resistant and -susceptible O. nubilalis larvae. Although a number of nucleotide differences were found in sequences from the Bt-resistant and -susceptible strains, the differences were not consistent with reduced trypsin-like activity in the Bt-resistant strain. However, the mRNA levels of OnT23 in the resistant strain were 2.7- and 3.8-fold lower than those of the susceptible strain as determined by northern blotting and real-time quantitative PCR, respectively. Thus, reduced trypsin-like activity may be attributed to reduced expression of OnT23 in Bt-resistant O. nubilalis. Our study provides new insights into Bt resistance management strategies, as resistance mediated by reduced Bt protoxin activation would be ineffective if resistant insects ingest a fully activated form of Cry1Ab toxin, either in spray formulations or transgenic Bt crops.