Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption. Received: 1 December 1995 / Accepted: 3 January 1996     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

The Crystal Proteins from Bacillus thuringiensis subsp. thompsoni Display a Synergistic Activity Against the Codling Moth, Cydia pomonella

Crystal proteins from Bacillus thuringiensis subsp. thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards. Inclusion bodies purified from strain HnC displayed an LC₅₀ of 3.34 × 10⁻³μg/μl. HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab. The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically. The toxicity of crystal proteins produced by the recombinant B. thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein. When the gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP. Received: 25 August 1999 / Accepted: 5 October 1999     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.     

A Cry1Ac Toxin Variant Generated by Directed Evolution has Enhanced Toxicity against Lepidopteran Insects

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.     

Cloning of Two New cry Genes from Bacillus thuringiensis subsp. wuhanensis Strain

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae. Received: 2 August 1999 / Accepted: 18 October 1999     

Aminopeptidase-N from the Helicoverpa armigera (Hubner) Brush Border Membrane Vesicles as a Receptor of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Brush border membrane vesicles (BBMVs) were prepared from the 2^nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn⁺⁺ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S. Received: 4 January 2001 / Accepted: 6 February 2001     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.     

Molecular and biological characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains for controlling tomato leafminer (<Emphasis Type="Italic">Tuta absoluta</Emphasis> Meyrick) (Lepidoptera: Gelechiidae) in Colombia

Twenty-eight soil samples were obtained from open fields and greenhouses used for tomato cultivation in various regions of Colombia. For functional characterization, 99 Bacillus thuringiensis (Bt) strains were isolated and characterized by abundance and morphology of microscopic crystals, SDS–PAGE of protein extracts and M-PCR analyses of genes of the cry1 family, as well as for their insecticidal activity against Tuta absoluta second instar larvae. Native Bt strains had amorphous (5%), bi-pyramidal (27%), square (8%), spherical (38%) and triangular (22%) crystal forms. Based on the presence of 1–4 different crystal forms, 18 different profiles were established. The SDS–PAGE analyses of protein extracts established ten different strain groups based on their protein band weight and potential biological activity. The M-PCR technique identified 35 native Bt strains based on the presence of the 6 genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D, whose frequency of occurrence was 76, 26, 21, 35, 32 and 8.8%, respectively. Thirteen different PCR profiles were found in native Bt strains. Several gene combinations tended to co-occur with elevated frequency, such as the pairs cry1Ac/cry1C, cry1Ab/cry1Ac and cry1Ab/cry1B, for which Pearson correlation coefficients were 0.69, 0.52 and 0.54, respectively. Native strains ZBUJTL39 and ZCUJTL11 had up to three times higher biological activity against T. absoluta second instar larvae than the reference strain Bt var. kurstaki HD1, with an LD₅₀ of 2.4 μg/ml (P < 0.05) for native Bt strain ZCUJTL11. This study suggests a high biodiversity of native Bt strains from tomato growing regions in Colombia, which has important implications for designing biological control strategies for T. absoluta.     

Southern analysis of BT-R 1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R₁ (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R ₁ , the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R₁ as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R₁. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R₁, whereas the other is a closely related homologue. Received: 27 March 1997 / Accepted: 30 June 1997     

Evidence of the Involvement of E358, A498 and C571 of a New Cry1Ac δ-endotoxin of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in its High Insecticidal Activity Against <Emphasis Type="Italic">Ephestia kuehniella</Emphasis>

A new cry1Ac-type gene was cloned from Bacillus thuringiensis strain BLB1, sequenced and expressed. The deduced amino acid sequence of the polypeptide has a predicted molecular mass of 132.186 kDa. The amino acid sequence alignment of BLB1 Cry1Ac with those of the published ones showed that this is a new delta-endotoxin. When compared with Cry1Ac of Bacillus thuringiensis strain HD1, it was found that BLB1 Cry1Ac harbours three mutations: V358E localized in domain II and V498A and Y571C localized in domain III. When the BLB1 Cry1Ac toxin was expressed in an acrystalliferous strain of B. thuringiensis (HD1CryB), bipyramidal crystals were produced. The spore–crystal mixture of this recombinant strain was at least two-fold more active against larvae of the lepidopteran Ephestia kuehniella than that of the recombinant strain expressing Cry1Ac of HD1. The study of the structural effect of these mutations suggested that they may stabilize key regions involved in the binding of the domains II and III to insect receptors.     

Construction of a <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> genetically-engineered strain harbouring the secreted Cry1Ia delta-endotoxin in its crystal

Unlike other Bacillus thuringiensis Cry proteins, Cry1Ia does not form a crystal since it is a secreted delta-endotoxin. We have engineered a Cry1Iac chimeric protein by substituting the C-terminal part of Cry1Ia by the corresponding Cry1Ac part. When expressed in an acrystalliferous B. thuringiensis strain, Cry1Iac did not crystallize, but when expressed in the crystalliferous strain BNS3, the chimeric protein co-crystallized with the endogenous Cry1A delta-endotoxins forming a typical bipyramidal crystal. The integration of Cry1Ia in the composition of the crystal of BNS3 led to an increase of its delta-endotoxin production (13%) and to an improvement (60%) of its toxicity against Agrotis ipsilon.     

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.     

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.     

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of <Emphasis Type="Italic">Adalia bipunctata</Emphasis> (Coleoptera: Coccinellidae): the importance of study design

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.