Showing papers on "Bacillus thuringiensis published in 2001"

Identification of a gene associated with Bt resistance in Heliothis virescens.

Abstract: Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt-resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon-mediated insertion was linked to high levels of resistance to the Bt toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele.

Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil

Abstract: There were no significant differences in the percent mortality and weight of earthworms (Lumbricus terrestris) after 40 days in soil planted with Bt (NK4640Bt) or non-Bt corn or after 45 days in soil amended with biomass of Bt or non-Bt corn. The toxin was present in the guts and casts of earthworms in soil planted with Bt corn or amended with biomass of Bt corn, but it was cleared within 2‐3 days from the guts after placing in fresh soil. There were no significant differences in the colony-forming units of culturable bacteria (including actinomycetes) and fungi and in the numbers of protozoa and nematodes between rhizosphere soil of Bt and non-Bt corn or between soil amended with biomass of Bt and non-Bt corn. The Cry1Ab protein in root exudates and biomass of Bt corn appears not to be toxic to earthworms, nematodes, protozoa, bacteria, and fungi. The presence of the toxin in the guts and casts of earthworms confirmed that the toxin released in root exudates and from transgenic biomass was bound on surface-active particles in soil, which protected the toxin from biodegradation, as has been observed in this laboratory with purified toxin. q 2001 Elsevier Science Ltd. All rights reserved.

Bt corn has a higher lignin content than non-Bt corn.

Abstract: Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33-97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis.

Abstract: Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen

Abstract: Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action

Abstract: The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects. These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut. There are two major classes, the cytolytic toxins and the δ-endotoxins. The former are produced by B. thuringiensis subspecies active on Diptera. The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects. They have a three-domain structure with extensive functional interactions among the domains. The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III. Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels. The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut. In this review, the mode of action of these δ-endotoxins will be discussed with emphasis on unique features.

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms

Abstract: Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

Bt Toxin Resistance from Loss of a Putative Carbohydrate-Modifying Enzyme

Abstract: The development of resistance is the main threat to the long-term use of toxins from Bacillus thuringiensis (Bt) in transgenic plants. Here we report the cloning of a Bt toxin resistance gene, Caenorhabditis elegans bre-5 , which encodes a putative β-1,3-galactosyltransferase. Lack of bre-5 in the intestine led to resistance to the Bt toxin Cry5B. Wild-type but not bre-5 mutant animals were found to uptake toxin into their gut cells, consistent with bre-5 mutants lacking toxin-binding sites on their apical gut. bre-5 mutants displayed resistance to Cry14A, a Bt toxin lethal to both nematodes and insects; this indicates that resistance by loss of carbohydrate modification is relevant to multiple Bt toxins.

Immunological analysis of phloem sap of Bacillus thuringiensis corn and of the nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab.

Abstract: Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B. thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA. Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes. Using this technique, no Cry1Ab was detected in the phloem sap. In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer. This was probably because of Cry1Ab toxin released from damaged cells. When analysing apterous adults of Rhopalosiphum padi L. and their honeydew, no Cry1Ab could be detected. In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores. These results suggest that R. padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit. In consequence it is hypothesized that R. padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.

Impact of Bt Cottons Expressing One or Two Insecticidal Proteins of Bacillus thuringiensis Berliner on Growth and Survival of Noctuid (Lepidoptera) Larvae

Abstract: A series of laboratory assays were performed to compare the relative impact of commercial and experimental cultivars of cotton, Gossypium hirsutum (L.), expressing zero, one, or two insecticidal proteins of Bacillus thuringiensis Berliner, on several lepidopteran pests. Assays in which larvae were fed fresh plant tissue indicated that dual-toxin B. thuringiensis (Bt) cultivars, expressing both Cry1Ac and Cry2Ab endotoxins of B. thuringiensis, were more toxic to bollworms, Helicoverpa zea (Boddie), fall armyworms, Spodoptera frugiperda (J. E. Smith), and beet armyworms, Spodoptera exigua (Hubner), than single-toxin cultivars expressing Cry1Ac. Assays in which lyophilized plant tissue was incorporated into artificial diet also indicated improved activity of the dual-toxin Bt cultivar compared with single-toxin plants. Both bollworm and tobacco budworm, Heliothis virescens (F.), growth was reduced by Bt cotton, particularly the dual-toxin cultivar. Although assays with lyophilized tissues were done using largely sublethal doses, bollworm survival was reduced by the dual-toxin cultivar. It appears that this newly developed Bt cotton expressing two toxins will be more effective and have a wider range of activity on these lepidopteran pests.

Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis.

Abstract: The coleopteran-active δ-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the δ-­endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an Rfree of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64–294); II, a three-sheet domain (residues 295–502); and III, a β-sandwich domain (residues 503–652). The monomers in the orthorhombic C2221 crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two δ-endotoxins.

Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions

Abstract: The widespread planting of corn genetically modified to produce Bacillus thuringiensis endotoxin has led to speculation that pollen from these fields might adversely affect nearby nontarget lepidopterans. A previous study of Bt corn engineered with Monsanto event 810 failed to detect an effect of pollen exposure on the black swallowtail, Papilio polyxenes, in either the field or the laboratory. Here, we report results of a field study investigating the impact of exposure to pollen from a Bt corn hybrid containing Novartis event 176 on two species of Lepidoptera, black swallowtails and monarch butterflies, Danaus plexippus. Nearly half of the 600 monarch larvae died within the first 24 h; this and subsequent mortality was not associated with proximity to Bt corn and may have been due in part to predation. Survivorship of black swallowtails was much higher than that of the monarchs and was also independent of proximity to the transgenic corn. However, despite five rainfall events that removed much of the pollen from the leaves of their host plants during the experiment, we observed a significant reduction in growth rates of black swallowtail larvae that was likely caused by pollen exposure. These results suggest that Bt corn incorporating event 176 can have adverse sublethal effects on black swallowtails in the field and underscore the importance of event selection in reducing environmental impacts of transgenic plants.

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil

Abstract: Aims: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. Methods and Results: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11·6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98°C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. Conclusions: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. Significance and Impact of the Study: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.

Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

Abstract: Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hubner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P 0.05). Single and dual-toxin genotypes remained superior (P 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed.

Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence

Abstract: PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini-Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini-Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini-Tn10 transposons mapped in a five-gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini-Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a DeltaplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum-sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in DeltaoppB, Deltaspo0A and DeltaoppB Deltaspo0A mutants indicates that Opp is required for plcR expression via a Spo0A-independent mechanism.

Evaluation of bacterial isolates from salty soils and bacillus thuringiensis strains for the biocontrol of fusarium dry rot of potato tubers

Abstract: SUMMARY A total of 83 spore-forming bacteria belonging to the genus Bacillus was isolated from Tunisian salty soils. These isolates as well as five additional strains of Bacillus thuringiensis, previously selected for their efficiency against insects, were tested in vitro and in vivo against Fusarium roseum var. sambucinum, the causal agent of dry rot of potato tubers. Results of the in vitro dual culture screening revealed that more than 50% of Bacillus spp. isolated from salty soils inhibited the growth of the pathogen in vitro. By contrast, all five B. thuringiensis strains failed to inhibit the growth of the pathogen in vitro. On wounded potato tubers, the most effective isolates obtained from salty soils were X7, X9, X16, I32 and G7, with a percentage of dry rot reduction ranging from 66 to 89%. These effective Bacillus isolates were identified as belonging to one of the species B. cereus (X9, X16 and G7), B. lentimorbus (X7) or B. licheniformis (I32). Although ineffective in vitro, B. thuringiensis strains inhibited dry rot development in vivo, with percentage inhibition scores ranging from 41 to 52%. While Bacillus isolates selected from salty soils best inhibited dry rot development when applied as young cultures (24 h), B. thuringiensis strains generally performed better as older cultures (48-72 h). The cell-filtrates of Bacillus spp. were unable to inhibit the growth of Fusarium. By contrast, volatiles liberated by the antagonists seem to contribute to the inhibition of the pathogen. The two isolates X16 of B. cereus and I32 of B. licheniformis as well as all 3 tested strains of B. thuringiensis (1T, 10T and 55T) were able to degrade colloidal chitin. Our experiments with the chromogenic chito-oligosaccharides indicated also that B. cereus (X16) and B. thuringiensis (55T) are able to produce N-acetyl-β-D-glucosaminidases, chitobiosidases and endochitinases. The isolate X16 of B. cereus consistently showed a chitinase activity 2 to 3 fold higher than that of strain 55T of B. thuringiensis. The hydrolysis of

Correlating Differences in Larval Survival and Development of Bollworm (Lepidoptera: Noctuidae) and Fall Armyworm (Lepidoptera: Noctuidae) to Differential Expression of Cry1A(c) δ-Endotoxin in Various Plant Parts Among Commercial Cultivars of Transgenic Bacillus thuringiensis Cotton

Abstract: Differences in larval survival and development of bollworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith), respectively, were found to exist among commercially available Cry1A(c) transgenic Bacillus thuringiensis Berliner (Bt) varieties. Using a quantification assay (ELISA) to measure the levels of delta-endotoxin in two of these varieties ('DP 451B/RR' and 'NuCOTN 33B'), differences in the amount of delta-endotoxin present in various plant parts was correlated with larval survival of bollworms and larval development of fall armyworms throughout the growing season. Larvae that were fed on DP 451B/RR completed development faster and exhibited better survivorship than those larvae fed NuCOTN 33B, whereas lower levels of delta-endotoxin were generally detected in plant parts from DP 451B/RR compared with NuCOTN 33B. These differences may impact population dynamics of these pests which may be a critical factor in managing resistance to Bt. Furthermore, the utility of using this system for providing information to the grower concerning varietal choices may be more common in the future.

Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite–humic acids–Al hydroxypolymers

Abstract: The equilibrium adsorption and binding of the active toxin from Bacillus thuringiensis subsp. kurstaki on complexes of montmorillonite‐ humic acids‐Al hydroxypolymers, as well as the biodegradation and the insecticidal activity of the bound toxin, were studied. Seventy percent of the total adsorption occurred within the first hour, and maximal adsorption occurred in ,8 h. Adsorption of the toxin on a constant amount of the complexes increased as the amount of the toxin added increased, and equilibrium adsorption isotherms of the L-type were obtained. There was essentially no desorption of the toxin after extensive washing of the toxin‐organomineral complexes with double distilled H2O and 1 M NaCl. The bound toxin was resistant to utilization by mixed microbial cultures from soil and to enzymatic degradation by Pronase E. Free and bound toxin were active against the larvae of Manduca sexta; the bound toxin retained the same activity after exposure to microbes or Pronase, whereas the toxicity of the free toxin decreased significantly. The results of these studies indicate that the release of transgenic plants and microorganisms expressing truncated genes that encode active insecticidal toxins from B. thuringiensis could result in the accumulation of these toxins in soil as a consequence of binding on surface-active soil particles. This persistence could pose a hazard to nontarget organisms, enhance the selection of toxin-resistant target species, and increase the control of target insect pests. q 2001 Elsevier Science Ltd. All rights reserved.

Overwintering Cost Associated with Resistance to Transgenic Cotton in the Pink Bollworm (Lepidoptera: Gelechiidae)

Abstract: Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent.

Genetically Engineered Organisms : Assessing Environmental and Human Health Effects

Abstract: Preface, B.E. Burrows Variability and Uncertainty in Crop-to-wild Hybridization, T. Klinger Factors Affecting the Spread of Resistant Arabidopsis thaliana Populations, J. Bergelson and C.B. Purrington Bt Crops: Benefits, Risks, and Non-target Effects on Wild Relatives, D.K. Letourneau, J.A. Hagen, and G.S. Robinson Resisting Resistance to Bt Corn, D.A. Andow Ecological Risks of Transgenic Virus-resistant Crops, A.G. Power Impacts of Genetically-engineered Crops on Non-target Herbivores: Bt-corn and Monarch Butterflies as a Case Study, J. Losey, J. Obrycki, and R.A. Hufbauer Transgenic Host Plant Resistance and Non-target Effects, A. Hilbeck Release, Persistence, and Biological Activity in Soil of Insecticidal Proteins from Bacillus thuringiensis, G. Stotsky Survival, Persistence, Transfer: The Fate of Genetically Modified Microorganisms and Recombinant DNA in Different Environments, B. Tappeser, M. Jager, and C. Eckelkamp The Spread of Genetic Constructs in Natural Insect Populations, H.R. Braig and G. Yan Ecological and Community Considerations in Engineering Arthropods to Suppress Vector-borne Disease, A. Spielman, J.C. Beier, and A.E. Kiszewski Environmental Risks of Genetically Engineered Vaccines, T. Traavik Methods to Assess Ecological Risks of Transgenic Fish Releases, W,M. Muir and R.D Howard Controversies in Designing Useful Ecological Assessments of Genetically Engineered Organisms, A.R. Kapuscinski

Chitinase from Bacillus thuringiensis subsp. pakistani

Abstract: The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.

Effects of Bt Cotton and Cry1Ac Toxin on Survival and Development of Pink Bollworm (Lepidoptera: Gelechiidae)

Abstract: We evaluated the effects of Bacillus thuringiensis (Bt) toxin Cry1Ac on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinophora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in Cry1Ac concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces Cry1Ac (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.

Fitness Costs and Maternal Effects Associated with Resistance to Transgenic Cotton in the Pink Bollworm (Lepidoptera: Gelechiidae)

Abstract: Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm, Perctinophora gossypiella (Saunders). We compared performance of pink bollworm strains resistant to Bt cotton with performance of their susceptible counterparts on non-Bt cotton. We found fitness costs that reduced survival on non-Bt cotton by an average of 51.5% in two resistant strains relative to the susceptible strains. The survival cost was recessive in one set of crosses between a resistant strain and the susceptible strain from which it was derived. However, crosses involving an unrelated resistant and susceptible strain indicated that the survival cost could be dominant. Development time on non-Bt cotton did not differ between the two related resistant and susceptible strains. A slight recessive cost affecting development time was suggested by comparison of the unrelated resistant and susceptible strains. Maternal effects transmitted by parents that had eaten Bt-treated artificial diet as larvae had negative effects on embryogenesis, adult fertility, or both, and reduced the ability of neonates to enter cotton bolls. These results provide further evidence that fitness costs associated with the evolution of resistance to Bt cotton are substantial in the pink bollworm.

Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth.

Abstract: Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis. Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin. Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites. Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca. three-fold-lower R(t) compared with the 688(s) colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.

Effect of Thiamethoxam on Entomopathogenic Microorganisms

Abstract: The compatibility of entomopathogenic microorganisms with thiamethoxam and other insecticides was studied in vitro and under field conditions. The microorganisms tested were: Aschersonia aleyrodis, Bacillus thuringiensis, Baculovirus anticarsia (NPVAg), Beauveria bassiana, Hirsutella thompsonii, Metarhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, Sporothrix insectorum and Verticillium lecanii. Two concentrations of each product were tested in the laboratory, based on the maximum and minimum recommended rates for application in the field. The products were added to specific culture medium for growth of the entomopathogens. Reproductive and vegetative growth were evaluated for fungi, and colony forming units (CFU) were evaluated for bacteria. For the field test, CFUs were considered for both fungi and bacteria and caterpillar mortality for the NPV of Anticarsia gemmatalis (Hueb.). Results showed that: (1) the action of the pesticides on the vegetative growth and sporulation of the microorganisms varied as a function of the chemical nature of the products, its concentration and the microbial species; (2) thiamethoxam was compatible with all microorganisms studied; (3) endosulfan, monocrotophos and deltamethrin were the insecticides that most affected B. thuringiensis, B. bassiana, M. anisopliae and S. insectorum; (4) thiamethoxam did not affect the inoculum potential of B. thuringiensis, B. bassiana or M. anisopliae when applied to bean crop (Phaseolus vulgaris); and (5) thiamethoxam did not affect the efficiency of the nuclear polyhedral virus of A. gemmatalis.

Life history traits of Helicoverpa zea (Lepidoptera: Noctuidae) on non-Bt and Bt transgenic corn hybrids in eastern North Carolina

Abstract: Transgenic varieties of field corn that express the Cry1Ab B. thuringiensis (Bt) toxin in ear tissue present the potential of reducing ear feeding by the corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae), and for reducing the size of populations of the insect infesting other host crops. Life history parameters of H. zea feeding on ears of conventional and Bt field corn varieties were measured in field plots in eastern North Carolina in 1997 and 1998. Transformation events investigated were Mon-810 and Bt-11. Bt corn was found to cause a steady mortality of larvae during development, but permitted ≈15–40% survival to the prepupal stage compared with non-Bt corn. Mortality of prepupae and pupae from Bt corn was also higher than from non-Bt corn, reducing overall adult production by 65–95%. The larvae that did survive grew more slowly on Bt than on non-Bt corn, and produced pupae that weighed 33% less. Pupation and adult eclosion were delayed by 6–10 d by feeding on Bt corn ears. Corn varieties...