Bt Proteins Research Articles

Trypsinized Cry1Fa and Vip3Aa have no detrimental effects on the adult green lacewing Chrysopa pallens (Neuroptera: Chrysopidae)

An exposure bioassay was established for green lacewing Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae) adults using a suitable artificial diet and honey–water solution (20%) to assess the toxicity of trypsinized Cry1Fa and Vip3Aa. Lethal and sub-lethal life table parameters were unaffected after C. pallens adults were given honey–water solutions containing Cry1Fa and Vip3Aa (50 µg/ml) and an artificial diet. In contrast, life table parameters of C. pallens adults were significantly affected when boric acid was mixed with the honey–water solution as a positive control. The uptake, temporal stability and bioactivity of Cry1Fa and Vip3Aa before and after C. pallens access to honey–water were confirmed using double antibody sandwich enzyme-linked immunosorbent assays and a bioactivity verification bioassay. The results prove that trypsinized Cry1Fa and Vip3Aa are safe for C. pallens adults, thus it is speculated that transgenic crops expressing Cry1Fa and Vip3Aa have no detrimental effects on lacewings and are compatible with biological control programs. This study describes a robust experimental design for evaluating the potential toxicity of alkaline gut-activated Bacillus thuringiensis (Bt) proteins on C. pallens adults which can be used to determine the potential toxicity of other Bt proteins on this species.

Evaluation of the Susceptibility Status of Spiny BollwormEarias biplaga(Walker) (Lepidoptera: Noctuidae) to Bt Cotton in South Africa

The bollworm complex of cotton in South Africa consists of the African bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), red bollworm, Diparopsis castanea (Hamps.) (Lepidoptera: Noctuidae), and the spiny bollworms, Earias biplaga (Walker) (Lepidoptera: Noctuidae) and Earias insulana (Boisduval) (Lepidoptera: Noctuidae) (Green et al. 2003). Larvae of these species are regarded as major pests of cotton since they tunnel into the growing tips, flowering buds and cotton bolls, resulting in damage and subsequent yield losses (Bennett 2015). Genetically modified (GM) transgenic Bt cotton plants that express Cry proteins have been cultivated in SouthAfrica since 1998 to control these lepidopteran pests (Thirtleet al. 2003). Bollgard®, expressing the Cry1Ac protein, was first commercially produced in South Africa in 1998 and discontinued after the 2010 growing season (ICAC 2007). Bollgard ll®, also registered for control of lepidopteran pests on cotton, is a stacked transgenic cotton which expresses two Bt proteins, namely Cry1Ac and Cry2Ab2 (Taverniers et al. 2008; Showalter et al. 2009).

Different response of an elite Bt restorer line of hybrid rice (Oryza sativa L.) in adaptation to nitrogen deficiency

Transgenic Bacillus thuringiensis (Bt) rice have been reported to acquire effective resistance against the target pests; however, the insertion and expression of alien Bt genes may have some unintended effects on the growth characteristics of rice. A screen-house experiment was conducted and repeated twice to investigate the growth characteristics and Bt protein expressions in two Bt rice lines [MH63 (Cry2A*) and MH63 (Cry1Ab/Ac)], which had different Bt protein expression levels in leaves, under zero nitrogen (N0) and recommended nitrogen (NR) fertilizer applications. Compared to the counterpart MH63, MH63 (Cry2A*) under N0 experienced accelerated leaf senescence and a lower internal N use efficiency (IEN), resulting in a 23.2% decrease in grain yield and a lower accumulated biomass. These variations were revealed to be correlated to the higher ratio of the Bt protein content to the soluble protein content (BTC/SPC) with a maximum value of 4.3‰ in MH63 (Cry2A*) leaves in the late growth stage. Under NR, no differences in growth characteristics between MH63 (Cry2A*) and MH63 were found. The growth characteristics of MH63 (Cry1Ab/Ac), with a lower BTC/SPC in the late growth stage compared to MH63 (Cry2A*), were identical to those of MH63 under the two N applications. Results show that the transgenic Bt rice MH63 (Cry2A*), with a relatively higher Bt protein expression in the late growth stage, had an inferior adaptation to nitrogen deficiency compared to its non-Bt counterpart. And this inferior adaptation was found to be correlated with the higher BTC/SPC in MH63 (Cry2A*) leaves in the late growth stage.

Does Bt rice pose risks to non-target arthropods? Results of a meta-analysis in China.

SummaryTransgenic Bt rice expressing the insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) has been developed since 1989. Their ecological risks towards non‐target organisms have been investigated; however, these studies were conducted individually, yielding uncertainty regarding potential agroecological risks associated with large‐scale deployment of Bt rice lines. Here, we developed a meta‐analysis of the existing literature to synthesize current knowledge of the impacts of Bt rice on functional arthropod guilds, including herbivores, predators, parasitoids and detritivores in laboratory and field studies. Laboratory results indicate Bt rice did not influence survival rate and developmental duration of herbivores, although exposure to Bt rice led to reduced egg laying, which correctly predicted their reduced abundance in Bt rice agroecosystems. Similarly, consuming prey exposed to Bt protein did not influence survival, development or fecundity of predators, indicating constant abundances of predators in Bt rice fields. Compared to control agroecosystems, parasitoid populations decreased slightly in Bt rice cropping systems, while detritivores increased. We draw two inferences. One, laboratory studies of Bt rice showing effects on ecological functional groups are mainly either consistent with or more conservative than results of field studies, and two, Bt rice will pose negligible risks to the non‐target functional guilds in future large‐scale Bt rice agroecosystems in China.

Review: biosafety assessment of Bt rice and other Bt crops using spiders as example for non-target arthropods in China.

Since the birth of transgenic crops expressing Bacillus thuringiensis (Bt) toxin for pest control, the public debate regarding ecological and environmental risks as well as benefits of Bt crops has continued unabated. The impact of Bt crops, especially on non-target invertebrates, has received particular attention. In this review, we summarize and analyze evidences for non-target effects of Bt rice on spiders, major predators in rice fields. Bt rice has been genetically modified to express the Bt protein, which has been shown to be transferred and accumulate in spiders as part of their food chain. Moreover, the Bt protein exhibits unintended effects on the physiology of spiders and spreads to higher trophic levels. Spiders possess unique physiological and ecological characteristics, revealing traits of surrogate species, and are thus considered to be excellent non-target arthropod model systems for study of Bt protein impacts. Due to the complexities of Bt protein transfer and accumulation mechanisms, as well as the apparent lack of information about resulting physiological, biochemical, and ecological effects on spiders, we raise questions and provide recommendations for promising further research.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

A laboratory assessment of the potential effect of Cry1Ab/Cry2Aj-containing Bt maize pollen on Folsomia candida by toxicological and biochemical analyses

The common soil arthropod Folsomia candida can survive well when fed only maize pollen and thus may be exposed to insecticidal proteins by ingesting insect-resistant genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins when being released into the soil. Laboratory experiments were conducted to assess the potential effects of Cry1Ab/Cry2Aj-producing transgenic Bt maize (Shuangkang 12–5) pollen on F. candida fitness. Survival, development, and the reproduction were not significantly reduced when F. candida fed on Bt maize pollen rather than on non-Bt maize pollen, but these parameters were significantly reduced when F. candida fed on non-Bt maize pollen containing the protease inhibitor E-64 at 75 μg/g pollen. The intrinsic rate of increase (rm) was not significantly reduced when F. candida fed on Bt maize pollen but was significantly reduced when F. candida fed on non-Bt maize pollen containing E−64. The activities of antioxidant-related enzymes in F. candida were not significantly affected when F. candida fed on Bt maize pollen but were significantly increased when F. candida fed on non-Bt pollen containing E−64. The results demonstrate that consumption of Bt maize pollen containing Cry1Ab/Cry2Aj has no lethal or sublethal effects on F. candida.

Cry3Bb1-Resistant Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Does Not Exhibit Cross-Resistance to DvSnf7 dsRNA.

Background and MethodologyThere is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination.Principal Findings and ConclusionsThe Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations.

Comparative Study of &amp;lt;i&amp;gt;Helicoverpa armigera&amp;lt;/i&amp;gt; (Hubner) (Lepidoptera: Noctuidae) Preference for Bt and Non-Bt Soybean Cultivars

Studies on the behavior of Helicoverpa armigera in relation to soybean cultivars expressing Bt proteins are fundamentally important for IPM. We determine the no-choice and feeding choice of H. armigera, the no-choice and oviposition choice in different Bt and non-Bt soybeans cultivars. In the first step it was carried out the experiment to determine the no-choice and food choice in test for 12 Bt and non-Bt soybean cultivars. Simultaneously the consumption was determined. In a second step it was evaluated the no-choice and oviposition choice in different soybean cultivars. Helicoverpa armigera caterpillars showed neither attractiveness and preference for food nor attractiveness and oviposition preference for Bt and non-Bt soybean leaves when simultaneously contrasting the tested cultivars. This study showed that Bt toxin did not influence the oviposition preference, and H. armigera adults did not differentiate Bt and non-Bt soybean cultivars for oviposition indicating no behavioral preference. Third instar caterpillars showed no discrimination between Bt and non-Bt soybean leaves. Research in this area must focus on the possibility of widespread planting of genetically modified soybeans containing the Bt protein, which is associated with selection pressure and the behavior of pest species in relation to their hosts, as well as on the adequacy of management tactics that is able to prevent the loss of technology efficiency.

Effects of Waterlogging on Bt Protein Content and Nitrogen Metabolism in Square of Bt Cotton

Effects of Waterlogging on Bt Protein Content and Nitrogen Metabolism in Square of Bt Cotton

Construction of high-efficiency transformation vector with multiple insect-resistant genes and expression in tobacco

A high-efficiency plant transgenic vector expressing multiple genes has been modified from expression vector pCAMBIA1302. Briefly, multiple cloning sites of a cloning vector pUCm-T were constructed into plant transgenic vector pCAMBIA1302 backbone using Bsp120 I and Spe I restriction digest site. Two Bt genes, cry1Ac and cry3A, were then constructed into the modified transgenic vector in different orders as p1870-35S::cry1Ac-Coy::cry3A and p1870-Coy::cry3A-35S::cry1Ac, respectively. Transgenic tobacco plants were generated using Agrobacterium-mediated transformation method. Polymerase chain reaction (PCR) results showed that both the exogenous genes were integrated into the genome of tobacco. Fluorescence qRT-PCR detected both transgenes and further ELISA assay validated the expressed Bt proteins. Transgenic lines showed enhanced resistance to larvae of Helicoverpa armigera Hubner in the further feeding assays. In conclusion, our modified transgenic vector was suitable for multiple gene expression and would greatly facilitate our future research.

Baseline susceptibility of different European lepidopteran and coleopteran pests to Bt proteins expressed in Bt maize: a systematic review

Abstract Background Lepidopteran and coleopteran species are the most important pests in maize. They can be controlled using genetically modified (GM) crops expressing insecticidal Bacillus thuringiensis (Bt) proteins. The long-term success of this technology demands a pest resistance management. Important information for the successful management of resistance is the baseline susceptibility of the different target pests to the different Bt proteins. The data on baseline susceptibility should enable risk assessors and managers to assess whether a GM maize produces a Bt protein in a high-dose to specific target organisms and resistance has evolved during the commercial cultivation of this GM maize events. Methods Our systematic search followed an a priori protocol including the database platforms Web of Science, Scopus, CAB abstracts, Science Direct and JSTOR. We additionally conducted a Google scholar search. We collated all search results and screened all retrieved articles using predetermined inclusion criteria. We identified 30 studies, which fulfilled the criteria of including a relevant Bt protein, a relevant species, an appropriate endpoint, and field-derived pest generations reared in the laboratory no longer than three generations. We then made a quality assessment to discover if the studies considered the dose response curves with confidence intervals, described the protein source, tested the protein concentration and the protein activity via positive controls, use more than ten larvae per concentration, more than two replications, and more than five protein concentrations. Since no quantitative synthesis was possible the synthesis of the results was done in a narrative form. Results Seventy percent of the studies fulfilled five or more criteria and 17% fulfilled four of the seven criteria. Six Bt proteins were tested on one or more of the four species Ostrinia nubilalis, Helicoverpa armigera, Sesamia nonagrioides, and Diabrotica virgifera virgifera. We extracted the baseline susceptibility for the given protein-species-combinations and the test method with the Bt protein applied either on the surface of the diet or incorporated. Although, the data displays a high heterogeneity and are thus hard to compare, they give an overview of the baseline susceptibility of lepidopteran/coleopteran pests to Bt proteins. Conclusion Our systematic review illustrates the heterogeneity of the data and indicates the necessity of standard protocols for testing susceptibility of insect pests, which provide comparable data. The cultivation of Bt crops, as with any other plant protection measure, is likely to result in resistance evolution in the target pests. Industry, policy makers, and research should combine knowledge to protect the benefits of this technology.

Battle in the New World: Helicoverpa armigera versus Helicoverpa zea (Lepidoptera: Noctuidae).

The corn earworm Helicoverpa zea (Boddie) and the old world bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) are allopatric species and occur in important agricultural crops. In maize, both species tend to infest the ear. The introduction of H. armigera in Brazil has created a new scenario, where these Helicoverpa species might cohabit and interact with one another, affecting the prevalence of each species in the agroecosystem, integrated pest management, and insect resistance management. In this study, larval occurrence and proportion of these species in maize was assessed in three regions of Brazil during three crop seasons. Interaction between the species was evaluated in interspecific and intraspecific scenarios under laboratory and field conditions. Helicoverpa zea was predominant in Rio Grande do Sul and the Planaltina, DF (central Brazil). In western Bahia, H. zea was predominant in the first collection, but approximately equal in number to H armigera in the second crop season. Both species exhibit high cannibalism/predation rates, and larval size was the primary factor for larval survival in the interaction studies. Larva of H. zea had higher survival when interacting with H. armigera, indicating that H. zea has an advantage in intraguild interactions with H. armigera in maize. Overall, the results from this study indicate that maize might play a role as a source of infestation or a sink of insecticide or Bt protein unselected H. armigera populations, depending on the H. zea:H. armigera intraguild competition and adult movement in the landscape.

Effect of Bt rice straw returning in soil on the growth and reproduction of Eisenia fetida.

Bacillus thuringiensis (Bt) protein can enter the soil through Bt crops straw returning to field, which may affect the growth and reproduction of soil animals, such as earthworms. Here, Bt rice (b2B138) and conventional rice (Anfeng A) straw were returned in soil to evaluate the impact of Bt rice on Eisenia fetida. Two varieties of rice straw were added into soil to breed E. fetida at the rates of 2.5%, 5%, 7.5% and 10%. The survival rate, relative growth rate, reproduction of earthworm, the Cry1Ab content in soil-straw mixture and earthworm were detected after 7, 15, 30, 45, 60, 75, 90 d. The results showed that Bt rice straw returning at higher concentrations (7.5% and 10%) inhibited the survival rate of E. fetida. Bt rice straw returning had no adverse effect on relative growth rate (RGR) of E. fetida. Bt rice straw treatment improved the reproduction of earthworms under 5%, 7.5% and 10% straw returning in soil. Enzyme-linked immunosorbent assay (ELISA) results indicated that immunoreactive Cry1Ab was detectable in soil-straw mixture and E. fetida from Bt rice treatments, and a strong decline was observed in soil-straw mixture with the increase of treated time. Therefore, Cry1Ab released from Bt rice straw returning at 2.5% and 5% concentration had no adverse effects on the growth and reproduction of E. fetida.

Performance of Cry1Ab‐susceptible and ‐heterozygous resistant populations of sugarcane borer in sequential feedings on non‐Bt and Bt maize plant tissue

AbstractA seed mix strategy has been used to provide refuge to susceptible insects for resistance management in planting transgenic maize expressing Bacillus thuringiensis Berliner (Bt) proteins. To determine whether larval movement in a seed mix planting creates favorable conditions for resistant heterozygotes of a target pest, performance of Cry1Ab‐susceptible (SS) and ‐heterozygous resistant (RS) populations of the sugarcane borer, Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), was evaluated in sequential feeding on non‐Bt and Cry1Ab (event MON 810) maize plant tissue. For each insect population, nine feeding sequences were employed. SS and RS feeding on non‐Bt plants for their entire larval stages survived well and &gt;60% of the adult pairs produced viable eggs, with an average of 269 progeny per female, whereas none of the two populations on Bt maize plants survived to the pupal stage. SS larvae could not develop to adults if the larvae fed on non‐Bt plants for ≤15 days and then moved to Bt plants. In contrast, 4.2–29.2% of RS larvae that fed on non‐Bt plants for ≥9 days and then moved to Bt plants developed to adults, and 63.6% of pairs of these adults produced viable eggs, with an average of 185 progeny per female. For SS larvae that fed on Bt plants for 1 or 2 days and then moved to non‐Bt plants, few larvae developed to adults with varied emergence times, whereas 28.1 and 13.5% RS larvae feeding on Bt plants for 1 and 2 days, respectively, successfully developed to adults; 43.8% of pairs of these adults produced viable eggs, with an average of 220 progeny per female. For the case of the single Bt gene maize plants (event MON 810), the results suggest that RS insects may have advantages in survival and reproduction over SS if RS larvae hatch and feed on Bt plants during the first 1 or 2 days and then move to non‐Bt plants. This advantage is less for RS larvae that hatch and feed on non‐Bt plants first and then move to Bt plants, unless the larval movement occurs in the later stages (e.g., fourth or fifth instars).

A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

A selective insecticidal protein from Pseudomonas for controlling corn rootworms.

The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant.

Cry1Ie is a kind of Bacillus thuringiensis (Bt) toxin protein which has a different action model than the Cry1Ab and Cry1Ac protein. The transgenic maize expressing Cry1Ie might be commercially used in the near future and it is urgent to develop a method to detect Cry1Ie protein in transgenic plants and their products. To develop an ELISA method, Cry1Ie protein was expressed in Escherichia coli strain Transetta DE3, purified with the Ni-NTA spin columns, and then validated by sequencing. Bioassay results showed that the purified Cry1Ie protein was highly toxic to the Asian corn borer. The polyclonal antibody (pAb) and the specific monoclonal antibody (mAb) 1G42D6 were generated from rabbit and mice which were immunized with Cry1Ie protein, respectively. Western blotting of crude Cry1Ie protein extracts was established by employing mAb 1G42D6, whereas the mAb 1G42D6 negligibly recognized other Bt proteins. Sandwich ELISA against Cry1Ie protein was established by coating with pAb and detecting with mAb 1G42D6. The limit of detection (LOD), the limit of quantification (LOQ), and the quantification range of the assay in different matrices of maize plant were determined as 0.27-0.51, 0.29-0.78, and 0.45-15.71ng/mL, respectively. Recoveries of Cry1Ie protein spiked in different maize tissues ranged from 75.1 to 99.5%. The established sandwich ELISA was verified using transgenic maize overexpressing Cry1Ie. The results in this study suggested that the established ELISA method is effective for detecting Cry1Ie protein in transgenic plants.

High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil.

The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL−1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.

Genetic Basis of Cry1F-Resistance in a Laboratory Selected Asian Corn Borer Strain and Its Cross-Resistance to Other Bacillus thuringiensis Toxins.

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive insect pest of corn in China. Susceptibility to the Cry1F toxin derived from Bacillus thuringiensis has been demonstrated for ACB, suggesting the potential for Cry1F inclusion as part of an insect pest management program. Insects can develop resistance to Cry toxins, which threatens the development and use of Bt formulations and Bt crops in the field. To determine possible resistance mechanisms to Cry1F, a Cry1F-resistant colony of ACB (ACB-FR) that exhibited more than 1700-fold resistance was established through selection experiments after 49 generations of selection under laboratory conditions. The ACB-FR strain showed moderate cross-resistance to Cry1Ab and Cry1Ac of 22.8- and 26.9-fold, respectively, marginally cross-resistance to Cry1Ah (3.7-fold), and no cross-resistance to Cry1Ie (0.6-fold). The bioassay responses of progeny from reciprocal F1 crosses to different Cry1 toxin concentrations indicated that the resistance trait to Cry1Ab, Cry1Ac and Cry1F has autosomal inheritance with no maternal effect or sex linked. The effective dominance (h) of F1 offspring was calculated at different concentrations of Cry1F, showing that h decreased as concentration of Cry1F increased. Finally, the analysis of actual and expected mortality of the progeny from a backcross (F1 × resistant strain) indicated that the inheritance of the resistance to Cry1F in ACB-FR was due to more than one locus. The present study provides an understanding of the genetic basis of Cry1F resistance in ACB-FR and also shows that pyramiding Cry1F with Cry1Ah or Cry1Ie could be used as a strategy to delay the development of ACB resistance to Bt proteins.