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.     

Cry1Ab protein levels in phytophagous insects feeding on transgenic corn: implications for secondary exposure risk assessment

Enzyme-linked immunosorbent assays (ELISA) and bioassays were used to estimate levels of Cry1Ab protein in four species of phytophagous insects after feeding on transgenic Bt-corn plants expressing Cry1Ab protein or artificial diets containing Cry1Ab protein. The level of Cry1Ab in insects feeding on sources containing the Cry1Ab protein was uniformly low but varied with insect species as well as food source. For the corn leaf aphid, Rhopalosiphum maidis (Fitch), feeding on diet solutions containing Cry1Ab protein, the level of the protein in the aphid was 250–500 times less than the original levels in the diet, whereas no Cry1Ab was detected by ELISA in aphids feeding on transgenic Bt-Corn plants. For the lepidopteran insects, Ostrinia nubilalis (Hübner), Helicoverpa zea (Boddie), and Agrotis ipsilon (Hufnagel), levels of Cry1Ab in larvae varied significantly with feeding treatment. When feeding for 24 h on artificial diets containing 20 and 100 ppm of Cry1Ab, the level of Cry1Ab in the larvae was about 57 and 142 times lower, respectively, than the original protein level in the diet for O. nubilalis, 20 and 34 times lower for H. zea, and 10 to 14 times lower for A. ipsilon. Diet incorporation bioassays with a susceptible insect (first instar O. nubilalis) showed significant Cry1Ab bioactivity present within whole body tissues of R. maidis and O. nubilalis that had fed on diet containing a minimum of 20 ppm or higher concentrations (100 or 200 ppm) of Cry1Ab, but no significant bioactivity within the tissues of these insects after feeding on transgenic Bt-corn plants. The relevance of these findings to secondary exposure risk assessment for transgenic Bt crops is discussed.     

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with ¹²⁵I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution.     

Analyses of Cry1Ab Binding in Resistant and Susceptible Strains of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae)

Cry1Ab toxin binding analysis was performed to determine whether resistance in laboratory-selected Ostrinia nubilalis strains is associated with target site alteration. Brush border membrane vesicles were prepared using dissected midguts from late instars of susceptible and resistant strains (Europe-R and RSTT) of O. nubilalis. Immunoblot analysis indicated that three different proteins bound to Cry1Ab toxin and were recognized by an anticadherin serum. In a comparison of resistant and susceptible strains, reduced Cry1Ab binding was apparent for all three bands corresponding to cadherin-like proteins in the Europe-R strain, while reduced binding was apparent in only one band for the RSTT strain. Real-time analysis of Cry1Ab binding to gut receptors using surface plasmon resonance suggested slight differences in affinity in both resistant strains. Additional binding analysis was conducted using ¹²⁵I-labeled Cry1Ab, Cry1Ac, and Cry1Aa. Slight differences were again observed between the resistant and susceptible strains for Cry1Ab binding. However, when binding of ¹²⁵I-labeled Cry1Aa was tested, a 10-fold reduction in the concentration of binding sites was observed in the Europe-R strain. Expression of the O. nubilalis cadherin gene was similar in both the resistant and susceptible strains and did not account for differences in binding. In combination, the results of the present work suggest that differences in susceptibility to Cry1A toxins in the Europe-R strain of O. nubilalis are associated with altered receptor binding, although the precise nature of this mechanism is still uncertain.     

Effects of infection with Nosema pyrausta on survival and development of offspring of laboratory selected Bt-resistant and Bt-susceptible European corn borers

Infection with Nosema pyrausta Paillot lengthens developmental period of Bt-susceptible Ostrinia nubilalis (Hübner) to a similar extent as feeding on Cry1Ab-incorporated diet in Cry1Ab-resistant O. nubilalis, and these two factors combined lengthen developmental period further than either alone. Resistant O. nubilalis mating with infected susceptible, or infected resistant partners would produce partially- and fully-resistant offspring, respectively, infected with N. pyrausta. To investigate the impacts on the progeny of such matings, test crosses were set up to produce partially- and fully Cry1Ab-resistant O. nubilalis offspring transovarially infected and not infected with N. pyrausta, which were exposed to Cry1Ab toxin at doses of 0, 3, or 30 ng/cm² for 7 days. Transovarial infection with N. pyrausta significantly decreased 7 day survival of partially and fully-resistant O. nubilalis feeding on 30 ng/cm² Cry1Ab. In addition, N. pyrausta infection delayed larval development (as measured by weight) of partially- and fully-resistant O. nubilalis feeding on 3 and 30 ng/cm² Cry1Ab. Impacts of natural enemies on target pests may have the potential to impact evolution of resistance. N pyrausta-infected O. nubilalis are more strongly affected by feeding on Bt, and would be less likely to survive to adulthood to pass on resistance to the next generation. This indigenous microsporidium may work to delay evolution of resistance in O. nubilalis by lowering their ability to survive on Bt.     

Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

A new isolate (IS5056) of Bacillus thuringiensis subsp. thuringiensis that produces a novel variant of Cry1Ab, Cry1Ab21, was isolated from soil collected in northeastern Poland. Cry1Ab21 was composed of 1,155 amino acids and had a molecular mass of 130.5 kDa, and a single copy of the gene coding for this endotoxin was located on a ~75-kbp plasmid. When synthesized by the wild-type strain, Cry1Ab21 produced a unique, irregular, bipyramidal crystal whose long and short axes were both approximately 1 μm long, which gave it a cuboidal appearance in wet mount preparations. In diet incorporation bioassays, the 50% lethal concentrations of the crystal-spore complex were 16.9 and 29.7 μg ml⁻¹ for second- and fourth-instar larvae of the cabbage looper, Trichoplusia ni, respectively, but the isolate was essentially nontoxic to larvae of the beet armyworm, Spodoptera exigua. A bioassay of autoclaved spore-crystal preparations showed no evidence of β-exotoxin activity, indicating that toxicity was due primarily to Cry1Ab21. Studies of the pathogenesis of isolate IS5056 in second-instar larvae of T. ni showed that after larval death the bacterium colonized and subsequently sporulated extensively throughout the cadaver, suggesting that other bacteria inhabiting the midgut lumen played little if any role in mortality. As T. ni is among the most destructive pests of vegetable crops in North America and has developed resistance to B. thuringiensis, this new isolate may have applied value.     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Variability in the cadherin gene in an Ostrinia nubilalis strain selected for Cry1Ab resistance

Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701 bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect.     

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08–6.44 ng mL⁻¹ and the medium inhibition of control (IC₅₀) was 0.73 ng mL⁻¹. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%–96.6% and with a coefficient of variation (CV) among 2.0%–8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.     

Cross-resistance and mechanism of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia nubilalis

The cross-resistance spectrum and biochemical mechanism of resistance to the Bacillus thuringiensis Cry1Ab toxin was studied in a field-derived strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) that was further selected in the laboratory for high levels (>1000-fold) of resistance to Cry1Ab. The resistant strain exhibited high levels of cross-resistance to Cry1Ac and Cry1Aa but only low levels of cross-resistance (<4-fold) to Cry1F. In addition, there was no significant difference between the levels of resistance to full-length and trypsin-activated Cry1Ab protein. No differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Significantly reduced binding of radiolabeled Cry1Aa was observed in the resistant strain whereas binding of Cry1Ab and Cry1Ac was practically the same in both resistant and susceptible strains. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of Cry1A toxins to a common receptor, which is more clearly revealed by the binding assays using radiolabeled Cry1Aa.     

Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured ¹²⁵I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for ¹²⁵I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit ¹²⁵I-Cry1Ab binding to BBMV. Cry1F inhibited ¹²⁵I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.     

Video-tracking and On-plant Tests Show Cry1Ab Resistance Influences Behavior and Survival of Neonate <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> Following Exposure to Bt Maize

To examine how resistance to Bacillus thuringiensis (Bt) toxins influences movement and survival of European corn borer (Ostrinia nubilalis [Hübner]) neonates, the responses of Cry1Ab-resistant , -susceptible, and hybrid (F1) larvae were examined using two different techniques. First, using an automated video-tracking system, aspects of O. nubilalis movement were quantified in the presence of artificial diet incorporating 50% non-Bt or insect-resistant Cry1Ab maize tissue. Second, O. nubilalis dispersal and survival were measured 48–72 h after hatching on a Cry1Ab maize plant surrounded by two non-Bt maize plants. Video tracking indicated the presence of Cry1Ab tissue increased the total distance moved (m), time moving (%), and time away from the diet (%) for O. nubilalis while decreasing meander (degrees/cm). However, resistant larvae showed reduced movement and increased meander (≈localized searching) relative to susceptible or hybrid larvae on diet incorporating Cry1Ab tissue. Conversely, when placed onto Cry1Ab maize plants, resistant larvae were more likely than susceptible O. nubilalis to disperse onto adjacent non-Bt plants. The difference in on-plant dispersal seems to reflect greater survival after toxin exposure for resistant larvae rather than increased activity. These results suggest that simplified ‘Petri dish’ tests may not be predictive of larval movement among non-Bt and insect-resistant Bt maize plants. Because models of O. nubilalis resistance evolution incorporate various movement and survival parameters, improved data for on-plant behavior and survival of Bt- resistant , -susceptible, and hybrid larvae should help preserve the efficacy of transgenic insect-resistant maize.     

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC₅₀) were 0.727–0.892 μg/mL and detection limit (IC₁₀) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10⁷ M⁻¹. The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.     

Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. ¹²⁵I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.     

Ten years of MON 810 resistance monitoring of field populations of Ostrinia nubilalis in Europe

From 2005 to 2015, Ostrinia nubilalis were collected in the most important maize‐growing areas in Europe where MON 810 was cultivated. The susceptibility of these O. nubilalis collections to the Cry1Ab protein was determined using overlay bioassays and compared to that of reference (control) strains. Larvae that died or did not moult after 7 days were used to calculate a moulting inhibition concentration (MIC). Two different batches of Cry1Ab protein were used over the course of this study. Between 2005 and 2015, 145 collections of O. nubilalis from 14 areas were analysed. The Cry1Ab susceptibility of populations from different geographic regions differed only slightly across years. The greatest variability in the MIC₅₀ for field samples collected from 2005 to 2011 and tested with batch 1 was 6.6‐fold in 2006. For field‐collected O. nubilalis, the difference between MIC₅₀ values of the most susceptible and most tolerant samples was 13.1‐fold for this period. For samples collected in 2012–2015 and tested with batch 2, the greatest variability was 4.1‐fold in 2014. A diagnostic concentration (MIC₉₉) was calculated for batch 1 (48 ng/cm²) using the results from all the collections in 2005–2012. Bridging experiments indicated that the diagnostic concentration for batch 2 was 28 ng/cm². From 2006 onwards, no O. nubilalis reached the 2nd larval stage when the diagnostic concentration of either batch of Cry1Ab was used. Only one insect collected from Romania in 2012 and two insects collected as reference strain from Spain in 2015 survived exposure to a dosage of 20 ng/cm², and none of these larvae survived on MON 810 maize. Our results indicate that there has been no significant change in susceptibility to Cry1Ab in European populations of O. nubilalis over the period 2005–2015.     

Potential of the Bacillus thuringiensis Toxin Reservoir for the Control of Lobesia botrana (Lepidoptera: Tortricidae), a Major Pest of Grape Plants

The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.