The Cultivation of Bt Corn Producing Cry1Ac Toxins Does Not Adversely Affect Non-Target Arthropods

Transgenic corn producing Cry1Ac toxins from Bacillus thuringiensis (Bt) provides effective control of Asian corn borer, Ostrinia furnacalis (Guenée), and thus reduces insecticide applications. However, whether Bt corn exerts undesirable effects on non-target arthropods (NTAs) is still controversial. We conducted a 2-yr study in Shangzhuang Agricultural Experiment Station to assess the potential impact of Bt corn on field population density, biodiversity, community composition and structure of NTAs. On each sampling date, the total abundance, Shannon''s diversity index, Pielou''s evenness index and Simpson''s diversity index were not significantly affected by Bt corn as compared to non-Bt corn. The “sampling dates” had a significant effect on these indices, but no clear tendencies related to “Bt corn” or “sampling dates X corn variety” interaction were recorded. Principal response curve analysis of variance indicated that Bt corn did not alter the distribution of NTAs communities. Bray-Curtis dissimilarity and distance analysis showed that Cry1Ac toxin exposure did not increase community dissimilarities between Bt and non-Bt corn plots and that the evolution of non-target arthropod community was similar on the two corn varieties. The cultivation of Bt corn failed to show any detrimental evidence on the density of non-target herbivores, predators and parasitoids. The composition of herbivores, predators and parasitoids was identical in Bt and non-Bt corn plots. Taken together, results from the present work support that Bt corn producing Cry1Ac toxins does not adversely affect NTAs.     

Bio‐safety evaluation of Cry1Ac,Cry2Ab,Cry1Ca,Cry1F and Vip3Aa on Harmonia axyridis larvae

Dietary exposure studies are initial steps in environmental risk assessments of genetically engineered plants on non‐target organisms. These studies are conducted in the laboratory where surrogate species are exposed to purified and biologically active insecticidal compounds at higher concentrations than those expected to occur in transgenic crops foliage. Thus, dietary exposure (early tier) tests provide robust data needed to make general conclusions about the susceptibility of the surrogate species to the test substance. For this, we developed suitable artificial diet and used it to establish a dietary exposure test for assessing the toxicity of midgut‐active insecticidal compounds to the larvae of the Asian ladybird beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Using boric acid as a model compound, we validated the bioassay established for H. axyridis larvae. An artificial diet containing boric acid which negatively affected survival, development and adult weights was offered to larvae and indicated that the bioassay was able to detect toxic effects of insecticidal substances incorporated in diets. Using this dietary exposure test, environmental risk assessment of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and the non‐Cry protein Vip3Aa was evaluated by analysing pupation rates, adult emergence rates, 7‐day larval weights, and freshly emerged male and female weights among the toxin treatments and a pure artificial diet. These life‐table parameters did not vary among artificial diets containing 200 μg/g Bt proteins or pure artificial diet. In contrast, boric acid adversely affected all life‐table parameters. Thus on these bases, we concluded H. axyridis larvae are not sensitive to these Bt proteins expressed in genetically engineered crops.     

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%–50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%–30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.     

Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.     

Bt Rice Expressing Cry2Aa Does Not Harm Cyrtorhinus lividipennis,a Main Predator of the Nontarget Herbivore Nilapavarta lugens

T2A-1 is a newly developed transgenic rice that expresses a synthesized cry2Aa gene driven by the maize ubiquitin promoter. T2A-1 exhibits high resistance against lepidopteran pests of rice. The brown planthopper, Nilapavarta lugens (Stål), is a main nontarget sap-sucking insect pest of rice, and Cyrtorhinus lividipennis (Reuter) is the major predator of the eggs and young nymphs of planthoppers. As C. lividipennis may expose to the Cry2Aa protein via N. lugens, it is therefore essential to assess the potential effects of transgenic cry2Aa rice on this predator. In the present study, three experiments were conducted to evaluate the ecological risk of transgenic cry2Aa rice to C. lividipennis: (1) a direct feeding experiment in which C. lividipennis was fed an artificial diet containing Cry2Aa at the dose of 10-time higher than that it may encounter in the realistic field condition; (2) a tritrophic experiment in which the Cry2Aa protein was delivered to C. lividipennis indirectly through prey eggs or nymphs; (3) a realistic field experiment in which the population dynamics of C. lividipennis were investigated using vacuum-suction. Both direct exposure to elevated doses of the Cry2Aa protein and prey-mediated exposure to realistic doses of the protein did not result in significant detrimental effects on the development, survival, female ratio and body weight of C. lividipennis. No significant differences in population density and population dynamics were observed between C. lividipennis in transgenic cry2Aa and nontransgenic rice fields. It may be concluded that transgenic cry2Aa rice had no detrimental effects on C. lividipennis. This study represents the first report of an assessment continuum for the effects of transgenic cry2Aa rice on C. lividipennis.     

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.     

转Bt基因稻谷对印度谷螟生长发育的影响

为建立仓储阶段转Bt水稻安全性评价中靶标害虫抗性汰选研究体系,配制了含不同比例(70%,50%,30%,10%)转Bt基因(Cry1Ab/Cry1Abc)明辉63水稻谷粉(简称Bt谷粉)的人工饲料饲喂印度谷螟Plodia interpunctella(Hübner),测定其对1～3龄幼虫在72h内的急性毒力,及对印度谷螟种群生长发育的影响,并采用ELISA法检测转基因稻谷和末龄幼虫体内Bt蛋白含量。结果发现:4种比例人工饲料对幼虫的毒力作用均发生在取食48h后,72h后剂量效应明显。含Bt水稻较高比例的饲料对印度谷螟发育的负面效应明显:幼虫死亡率高,发育历期延长。Bt蛋白在幼虫体内含量与对应饲料中的含量基本成正比。综合考虑,将Bt杀虫蛋白含量2.35μg/g作为转Bt基因稻谷对印度谷螟的亚致死剂量最为合适。     

A Comprehensive Assessment of the Effects of Bt Cotton on Coleomegilla maculata Demonstrates No Detrimental Effects by Cry1Ac and Cry2Ab

The ladybird beetle, Coleomegilla maculata (DeGeer), is a common and abundant predator in many cropping systems. Its larvae and adults are predaceous, feeding on aphids, thrips, lepidopteran larvae and plant tissues, such as pollen. Therefore, this species is exposed to insecticidal proteins expressed in insect-resistant, genetically engineered cotton expressing Cry proteins derived from Bacillus thuringiensis (Bt). A tritrophic bioassay was conduced to evaluate the potential impact of Cry2Ab- and Cry1Ac-expressing cotton on fitness parameters of C. maculata using Bt-susceptible and -resistant larvae of Trichoplusia ni as prey. Coleomegilla maculata survival, development time, adult weight and fecundity were not different when they were fed with resistant T. ni larvae reared on either Bt or control cotton. To ensure that C. maculata were not sensitive to the tested Cry toxins independent from the plant background and to add certainty to the hazard assessment, C. maculata larvae were fed artificial diet incorporated with Cry2Ab, Cry1Ac or both at >10 times higher concentrations than in cotton tissue. Artificial diet containing E-64 was included as a positive control. No differences were detected in any life-table parameters between Cry protein-containing diet treatments and the control diet. In contrast, larvae of C. maculata fed the E-64 could not develop to the pupal stage and the 7-d larval weight was significantly negatively affected. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources were confirmed by ELISA and sensitive-insect bioassays. Our results show that C. maculata is not affected by Bt cotton and is not sensitive to Cry2Ab and Cry1Ac at concentrations exceeding the levels in Bt cotton, thus demonstrating that Bt cotton will pose a negligible risk to C. maculata. More importantly, this study demonstrates a comprehensive system for assessing the risk of genetically modified plants on non-target organisms.     

Capturing the interaction types of two Bt toxins Cry1Ac and Cry2Ab on suppressing the cotton bollworm by using multi‐exponential equations

Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis (Bt) toxin transgenic crops. Therefore, transgenic crops including two or more mixed Bt‐toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross‐resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi‐exponential equations (namely bi‐ and tri‐exponential equations) to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi‐exponential equations with the generalized linear model (GLM) in describing the combination effects, and found that the bi‐ and tri‐exponential equations are better than GLM. Moreover, the bi‐exponential equation can also provide the optimal dose combinations for two toxins.     

Prey-mediated effects of Cry1Ab toxin and protoxin and Cry2A protoxin on the predator Chrysoperla carnea

Laboratory feeding experiments were carried out to study prey-mediated effects of artificial diet containing Bacillus thuringiensis proteins on immature Chrysoperla carnea. Activated Cry1Ab toxin and the protoxins of Cry1Ab and Cry2A were mixed into standard meridic diet for Spodoptera littoralis (Boisduval) larvae at the following concentrations; for Cry1Ab toxin, 25, 50, 100 g g^–1 diet were used; for Cry1Ab protoxin, the concentration was doubled (50 g g^–1 diet, 100 g g^–1 diet and 200 g g^–1 diet) to give relative comparable levels of toxin concentration. Cry2A protoxin was incorporated into the meridic diet at one concentration only (100 g g^–1 diet). For the untreated control, the equivalent amount of double distilled water was added to the meridic diet. Individual C. carnea larvae were raised on S. littoralis larvae fed with one of the respective treated meridic diets described above. The objectives were to quantify and compare the resulting effects on mortality and development time of C. carnea with those observed in two previous studies investigating prey-mediated effects of transgenic Cry1Ab toxin-producing corn plants and the other studying effects of Cry1Ab toxin fed directly to C. carnea larvae. Mean total immature mortality for chrysopid larvae reared on B. thuringiensis-fed prey was always significantly higher than in the control (26%). Total immature mortality of C. carnea reared on Cry1Ab toxin 100 g g^–1 diet-fed prey was highest (78%) and declined with decreasing toxin concentration. Cry1Ab protoxin-exposed C. carnea larvae did not exhibit a dose response. Prey-mediated total mortality of Cry1Ab protoxin-exposed chrysopid larvae was intermediate (46–62%) to Cry1Ab toxin exposed (55–78%) and Cry2A protoxin (47%) exposed C. carnea. In agreement with the previous studies, total development time of C. carnea was not consistently, significantly affected by the Bt-treatments except at the highest Cry1Ab toxin concentration. However, both highest mortality and delayed development of immature C. carnea raised on Cry1Ab toxin 100 g g^–1 diet – fed prey may have been confounded with an increased intoxication of S. littoralis larvae that was observed at that concentration. At all other B. thuringiensis protein concentrations S. littoralis was not lethally affected. Comparative analysis of the results of this study with those of the two previous studies revealed that in addition to prey/herbivore by B. thuringiensis interactions, also prey/herbivore by plant interactions exist that contribute to the observed toxicity of B. thuringiensis – fed S. littoralis larvae for C. carnea. These findings demonstrate that tritrophic level studies are necessary to assess the long-term compatibility of insecticidal plants with important natural enemies.     

Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important lepidopteran pest of cotton (Gossypium spp.) in Australia and the Old World. From 2002, F2 screens were used to examine the frequency of resistance alleles in Australian populations of H. armigera to Bacillus thuringiensis (Bt) CrylAc and Cry2Ab, the two insecticidal proteins present in the transgenic cotton Bollgard II. At that time, Ingard (expressing Cry1Ac) cotton had been grown in Australia for seven seasons, and Bollgard II was about to be commercially released. The principal objective of our study was to determine whether sustained exposure caused an elevated frequency of alleles conferring resistance to Cry1Ac in a species with a track record of evolving resistance to conventional insecticides. No major alleles conferring resistance to Cry1Ac were found. The frequency of resistance alleles for Cry1Ac was <0.0003, with a 95% credibility interval between 0 and 0.0009. In contrast, alleles conferring resistance to Cry2Ab were found at a frequency of 0.0033 (0.0017, 0.0055). The first isolation of this allele was found before the widespread deployment of Bollgard II. For both toxins the experiment-wise detection probability was 94.4%. Our results suggest that alleles conferring resistance to Cry1Ac are rare and that a relatively high baseline frequency of alleles conferring resistance to Cry2Ab existed before the introduction of Bt cotton containing this toxin.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.     

Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green Lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae)

Adults of the common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), are prevalent pollen-consumers in maize fields. They are therefore exposed to insecticidal proteins expressed in the pollen of insect-resistant, genetically engineered maize varieties expressing Cry proteins derived from Bacillus thuringiensis (Bt). Laboratory experiments were conducted to evaluate the impact of Cry3Bb1 or Cry1Ab-expressing transgenic maize (MON 88017, Event Bt176) pollen on fitness parameters of adult C. carnea. Adults were fed pollen from Bt maize varieties or their corresponding near isolines together with sucrose solution for 28 days. Survival, pre-oviposition period, fecundity, fertility and dry weight were not different between Bt or non-Bt maize pollen treatments. In order to ensure that adults of C. carnea are not sensitive to the tested toxins independent from the plant background and to add certainty to the hazard assessment, adult C. carnea were fed with artificial diet containing purified Cry3Bb1 or Cry1Ab at about a 10 times higher concentration than in maize pollen. Artificial diet containing Galanthus nivalis agglutinin (GNA) was included as a positive control. No differences were found in any life-table parameter between Cry protein containing diet treatments and control diet. However, the pre-oviposition period, daily and total fecundity and dry weight of C. carnea were significantly negatively affected by GNA-feeding. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources as well as the uptake by C. carnea was confirmed. These results show that adults of C. carnea are not affected by Bt maize pollen and are not sensitive to Cry1Ab and Cry3Bb1 at concentrations exceeding the levels in pollen. Consequently, Bt maize pollen consumption will pose a negligible risk to adult C. carnea.     

Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate‐binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC₅₀) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC₅₀ = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.     

Biological Activity of Cry1Ab Toxin Expressed by Bt Maize Following Ingestion by Herbivorous Arthropods and Exposure of the Predator Chrysoperla carnea

A major concern regarding the deployment of insect resistant transgenic plants is their potential impact on non-target organisms, in particular on beneficial arthropods such as predators. To assess the risks that transgenic plants pose to predators, various experimental testing systems can be used. When using tritrophic studies, it is important to verify the actual exposure of the predator, i.e., the presence of biologically active toxin in the herbivorous arthropod (prey). We therefore investigated the uptake of Cry1Ab toxin by larvae of the green lacewing (Chrysoperla carnea (Stephens); Neuroptera: Chrysopidae) after consuming two Bt maize-fed herbivores (Tetranychus urticae Koch; Acarina: Tetranychidae and Spodoptera littoralis (Boisduval); Lepidoptera: Noctuidae) by means of an immunological test (ELISA) and the activity of the Cry1Ab toxin following ingestion by the herbivores. Moreover, we compared the activity of Cry1Ab toxin produced by Bt maize to that of purified toxin obtained from transformed Escherichia coli, which is recommended to be used in toxicity studies. The activity of the toxin was assessed by performing feeding bioassays with larvae of the European corn borer (Ostrinia nubilalis (Hübner); Lepidoptera: Crambidae), the target pest of Cry1Ab expressing maize. ELISA confirmed the ingestion of Bt toxin by C. carnea larvae when fed with either of the two prey species and feeding bioassays using the target pest showed that the biological activity of the Cry1Ab toxin is maintained after ingestion by both herbivore species. These findings are discussed in the context of previous risk assessment studies with C. carnea. The purified Cry1Ab protein was more toxic to O. nubilalis compared to the plant-derived Cry1Ab toxin when applied at equal concentrations according to ELISA measurements. Possible reasons for these findings are discussed.     

Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

The effects of Cry2Ab and Cry1Ac insecticidal proteins on protease activity in the midgut of Helicoverpa armigera

为明确Cry2Ab和Cry1Ac 2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera(Hübner)中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异.结果发现:Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6.67μg／g Cry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6.43倍.Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理.Cry2Ab和Cry1Ac 2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2.22μg/g混用时,处理12b后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照.     

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.