Characterizing indirect prey-quality mediated effects of a Bt crop on predatory larvae of the green lacewing, Chrysoperla carnea

There is increasing evidence that insecticidal transgenic crops can indirectly cause detrimental effects on arthropod predators or parasitoids when they prey on or parasitize sublethally affected herbivores. Our studies revealed that Chrysoperla carnea is negatively affected when fed Bt-susceptible but not Cry1Ac-resistant Helicoverpa armigera larvae that had fed Bt-transgenic cotton expressing Cry1Ac. This despite the fact that the predators ingested 3.5 times more Cry1Ac when consuming the resistant caterpillars. In order to detect potential differences in the nutrient composition of prey larvae, we evaluated the glycogen and lipid content plus the sugar and free amino acid content and composition of caterpillars fed non-Bt and Bt cotton. The only change in susceptible H. armigera larvae attributable to Bt cotton feeding were changes in sugar concentration and composition. In case of the Cry1Ac-resistant H. armigera strain, feeding on Bt cotton resulted in a reduced glycogen content in the caterpillars. The predators, however, appeared to compensate for the reduced carbohydrate content of the prey by increasing biomass uptake which caused an excess intake of the other analyzed nutritional compounds. Our study clearly proves that nutritional prey-quality factors other then the Bt protein underlie the observed negative effects when C. carnea larvae are fed with Bt cotton-fed prey. Possible factors were an altered sugar composition or fitness costs associated with the excess intake of other nutrients.     

Risk assessment system establishment for evaluating the potential impacts of imported Bacillus thuringiensis maize on a non-target insect,Tenebrio molitor

Large amounts of genetically modified grains producing Bacillus thuringiensis (Bt) toxins have been imported to Korea. Therefore, the establishment of a risk assessment system for evaluating the potential impacts of imported Bt maize on non-target insects is important. Before evaluating the environmental impacts of Bt grains of unknown origin, Cry protein types must first be identified in test Bt grains. Cry toxins of imported Bt maize grains were analyzed by ELISA. Because all tested Bt maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as the non-target insect species. A domestic maize strain that showed few differences in nutritional composition compared to the Bt maize grain was used as the alternative non-Bt control. Slightly increased survival rate and head capsule width of Bt maize-fed T. molitor were observed, indicating that Bt maize has no sub-chronic adverse effects on T. molitor. An ELISA test revealed that concentrations of Cry1A toxins slowly increased in the body of T. molitor when the insects were fed Bt maize. Such substantial amounts of Cry toxins remaining in the alimentary tract of larvae indicate that Cry toxins can be transferred to the higher trophic level of predatory insects. However, no Cry proteins were detected in the hemolymph of the Bt maize-fed larvae, suggesting that there is little possibility of Cry toxin exposure via T. molitor to the higher endoparasitoids. The risk assessment strategies and protocols established in this study may also be applicable to other imported Bt crops in Korea.     

Use of an artificial diet system to study the toxicity of gut-active insecticidal compounds on larvae of the green lacewing Chrysoperla sinica

A semi-liquid artificial diet was established and found to be a suitable food source for Chrysoperla sinica larvae, comparable to aphid prey. Using the artificial diet, we established and validated a dietary exposure assay by using the insecticidal potassium arsenate (PA) as positive control. Dose-dependent responses were documented for all observed life-table parameters of C. sinica larvae such as survival rate, pupation rate, larval weight, and larval development time. Thus, the dietary assay can detect the effects of insecticidal compounds on the survival and development of C. sinica larvae. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac, and Cry2Aa proteins (which are produced by transgenic maize, cotton or rice plants) to C. sinica larvae. Artificial diets containing Galanthus nivalis agglutinin (GNA) or PA were included as positive controls. Survival and development of C. sinica larvae were not affected when the artificial diet contained purified Cry1Ab, Cry1Ac, or Cry2Aa at 200 μg/g diet. In contrast, C. sinica larvae were adversely affected when the diet contained PA and GNA. The stability and bioactivity of the Cry proteins in the diet and Cry protein uptake by the lacewing larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on green lacewing larvae. The experiments with the Cry proteins demonstrate that C. sinica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry2Aa.     

Consumption of Bt Rice Pollen Containing Cry1C or Cry2A Protein Poses a Low to Negligible Risk to the Silkworm Bombyx mori (Lepidoptera: Bombyxidae)

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm² leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥150 grains/cm² leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori.     

Binding of Bacillus thuringiensis Cry1A toxins with brush border membrane vesicles of maize stem borer (Chilo partellus Swinhoe)

Maize stem borer (Chilo partellus) is a major insect pest of maize and sorghum in Asia and Africa. Bacillus thuringiensis (Bt) δ-endotoxins have been found effective against C. partellus, both in diet-overlay assay and in transgenic plants. Gene stacking as one of the resistance management strategies in Bt maize requires an understanding of receptor sharing and binding affinity of δ-endotoxins. In the present study, binding affinity of three fluorescein isothiocyanate labeled Cry1A toxins showed high correlation with the toxicity of respective δ-endotoxins. Competitive binding studies showed that Cry1Ab toxins share some of the binding sites with Cry1Aa and Cry1Ac with low affinity and that Cry1Ab may have additional binding sites that are unavailable to the other two toxins tested.     

Development of the predator Chrysoperla carnea (Stephens) as affected by feeding on Helicoverpa armigera (Hübner) reared on transgenic Cry 2Ab tomato plants

Investigations were conducted using Helicoverpa armigera fed on transgenic tomato plants containing a synthetic Cry2ab gene aiming to evaluate its impact on the development of the predator Chrysoperla carnea. Prey consumption and development parameters of C. carnea were assessed. No obvious differences were observed in the development of C. carnea that preyed on H. armigera fed on transgenic Bt tomato plants as compared to the control. This shows obvious that transgenic Bt tomato plants can be safely used as an efficient tool for the biocontrol of H. armigera with no effect on its predator, C. carnea.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

Effect of Bt maize on the reproduction and development of saprophagous Diptera over multiple generations

A laboratory experiment was used to quantify the effects of Bt maize on Drosophila melanogaster and Megaselia scalaris, representatives of two saprophagous dipteran families (Drosophilidae, Phoridae). Freshly hatched larvae were reared on a diet containing decaying maize leaves. Two transgenic maize varieties, expressing Cry3Bb1 or Cry1Ab, and their corresponding isolines were tested. In an additional treatment, a solution of pure Cry1Ab was added to the maize diet. According to quantitative ELISA analyses, all Bt diets and all larvae feeding on Bt maize contained low concentrations of Cry proteins but Cry proteins were not detected in adults, thus, predators of the larvae are exposed to Cry proteins whereas predators of adult flies are not. Highest concentrations were in larvae feeding on a maize diet supplemented with a Cry1Ab protein solution. The developmental time and fertility (offspring/female) were measured over four generations for D. melanogaster and over three generations for M. scalaris. Only a few significant differences were found between transgenic and non-transgenic treatments but the differences were not consistent and did not indicate any negative effects of Bt proteins. We conclude that D. melanogaster and M. scalaris larvae are not affected in the long term when feeding and developing on decaying Cry1Ab and Cry3Bb1 maize leaves.     

Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix ??-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix ??-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene.We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.     

Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees.     

A 3-year field-scale monitoring of foliage-dwelling spiders (Araneae) in transgenic Bt maize fields and adjacent field margins

Concerns have been raised that genetically modified Bt maize may harm non-target organisms, and there is a general call and need for a risk assessment of Bt maize. Spiders are important pest predators in agroecosystems and in maize, and can be exposed to the Bt toxin by herbivorous or pollen-collecting prey, by active Bt maize pollen feeding, and by ingesting their pollen-dusted webs. The foliage-dwelling spider fauna of Bt maize fields and adjacent margins was monitored and compared to non-transgenic maize fields. The study took place during the vegetation seasons of 2001–2003 in Bavaria, South Germany. Maize fields and adjacent nettle field margins were colonized by a typical spider assemblage, dominated by space-web spiders (Theridiidae and Linyphiidae). Abundance and species richness of spiders was higher in nettle margins than in maize fields. The proportion of hunting spiders tended to be higher in nettle margins, whereas space-web spiders tended to be more frequent in maize fields. Bt maize showed no consistent effect on individual numbers, species richness and guild structure of spiders in maize fields and adjacent nettle field margin strips. The spider abundance was higher in Bt treatments in 2003, whereas in 2001 and 2002 no significant differences were found. The results provide an important contribution for the implementation of case-specific and general surveillance of transgenic plants to be employed due to the regulations of the European Community.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

Bt-maize event MON?88017 expressing Cry3Bb1 does not cause harm to non-target organisms

This review paper explores whether the cultivation of the genetically modified Bt-maize transformation event MON?88017, expressing the insecticidal Cry3Bb1 protein against corn rootworms (Coleoptera: Chrysomelidae), causes adverse effects to non-target organisms (NTOs) and the ecological and anthropocentric functions they provide. Available data do not reveal adverse effects of Cry3Bb1 on various NTOs that are representative of potentially exposed taxonomic and functional groups, confirming that the insecticidal activity of the Cry3Bb1 protein is limited to species belonging to the coleopteran family of Chrysomelidae. The potential risk to non-target chrysomelid larvae ingesting maize MON?88017 pollen deposited on host plants is minimal, as their abundance in maize fields and the likelihood of encountering harmful amounts of pollen in and around maize MON?88017 fields are low. Non-target adult chrysomelids, which may occasionally feed on maize MON?88017 plants, are not expected to be affected due to the low activity of the Cry3Bb1 protein on adults. Impacts on NTOs caused by potential unintended changes in maize MON?88017 are not expected to occur, as no differences in composition, phenotypic characteristics and plant-NTO interactions were observed between maize MON?88017 and its near-isogenic line.     

Assessment of potential impacts due to unintentionally released Bt maize plants on non-target aphid Rhopalosiphum padi (Hemiptera: Aphididae)

Genetically modified maize crops expressing Bacillus thuringiensis (Bt) toxins (Bt maize) are increasingly cultivated worldwide, and large amounts of Bt maize have been imported to Korea. Before evaluating the environmental impacts of Bt maize of unknown origin on non-target insects, crystal (Cry) protein types in the imported Bt maize plants were identified. Because Cry1F was found in the tested Bt maize plants, Rhopalosiphum padi, a non-lepidopteran species, was selected as the non-target insect species. Additionally, a widely cultivated domestic maize strain was selected as an alternative control. No difference in survival rate, alata vivipara production, or host preference was observed between R. padi fed on the Bt maize and the control non-Bt maize, indicating that Bt maize plants had no sub-chronic adverse effects on R. padi. The average number of nymphs from Bt maize-fed aphids was 1.73-fold higher than that of non-Bt maize-fed aphids, implying that R. padi population density can increase after several generations in Bt maize fields. An enzyme-linked immunosorbent assay revealed that Cry1F toxin concentrations increased gradually in the body of R. padi when they were fed Bt maize, but that all ingested Cry toxins were excreted within 10 days after Bt-fed aphids were transferred to non-Bt maize, suggesting little possibility of Cry toxin exposure via R. padi to the endoparasitoids. However, the possibility still remains that Cry toxins can be transferred to predatory insects in higher trophic levels if they consume Bt maize-fed aphids.     

Assessing the utilization of a carbohydrate food source and the impact of insecticidal proteins on larvae of the green lacewing, Chrysoperla carnea

A concern with the widespread use of insecticidal transgenic crops is their potential to adversely affect non-target organisms, including biological control agents such as larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae). Since the insecticidal proteins expressed by the current transgenic plants are active only after ingestion, dietary bioassays are required to test direct effects on non-target organisms. After showing that C. carnea larvae utilize carbohydrate foods, we exposed them to insecticidal proteins dissolved in a sucrose solution. Feeding on snowdrop lectin (Galanthus nivalis agglutinin, GNA) as a model compound, the larvae were negatively affected in a number of life-table parameters. Interestingly, GNA caused a prolongation in first instar development, but had no effect on subsequent utilization of prey resulting in an increased weight of second instars. Comparable studies with avidin, a biotin-binding protein, revealed strong effects on C. carnea survival at the concentration tested. Despite the fact that the proteolytic digestion of C. carnea larvae is reported to be dominated by serine proteases, ingestion of soybean trypsin inhibitor (SBTI) did not cause any detrimental effects. Similarly, two Cry proteins derived from Bacillus thuringiensis (Cry1Ac and Cry1Ab) did not cause negative effects on C. carnea, what is consistent with earlier studies. The here presented bioassay provides a valuable tool to assess direct impacts of insecticidal proteins to C. carnea larvae and other predators that are known to feed on carbohydrate solutions.     

In vitro hydrolysis of Bacillus thuringiensis Cry1Ac toxin by gut proteases of Nilaparvata lugens (Stål) and binding assays of Cry1Ac toxin with brush border membrane of N. lugens midgut

Bacillus thuringiensis (Bt) and transgenic crops carrying cry genes are widely used in the management of lepidopteran and coleopteran pests. However, almost none of the Cry toxins have insecticidal properties against sap-sucking insects, such as planthoppers, leafhoppers and aphids. To understand the low insecticidal activity of Cry1Ac toxin on sap-sucking insects, we investigated two critical steps in the Bt-intoxication cascade: the proteolytic processing of Cry1Ac toxin by gut proteases, and the binding of Cry1Ac to brush border membrane vesicles (BBMV) of Nilaparvata lugens. Proteolytic processing of Cry1Ac protoxin by N. lugens gut proteases resulted in an ～65?kDa product, similar to the expected size of the trypsin-activated Cry1Ac toxin. In addition, activation of cysteine proteases in N. lugens gut increased the efficiency of proteolytic activities in the processing of Cry1Ac. However, feeding N. lugens nymphs with either Cry1Ac protoxin or trypsin-activated Cry1Ac toxin resulted in low mortalities. The LC₅₀ of Cry1Ac protoxin and trypsin-activated Cry1Ac was 198.92 and 450.18?μg/mL, respectively. In vitro binding analysis of BBMV with the pre-activated Cry1Ac showed that Cry1Ac toxin could specifically bind to the BBMV. However, binding competition with 500-fold molar excess GalNAc (N-acetyl-d-galactosamine) suggested that the binding was not mediated by GalNAc-like glycoproteins. These results indicate that Cry1Ac toxin could be successfully processed by the treatment of N. lugens gut proteases. However, the binding of Cry1Ac toxin to the midgut brush border membrane was not mediated by GalNAc-like glycoprotein. This may be responsible for the low susceptibility of N. lugens to Cry1Ac.     

Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties

Ribosomal 16S rRNA gene pyrosequencing was used to explore whether the genetically modified (GM) Bt-maize hybrid MON 89034 × MON 88017, expressing three insecticidal recombinant Cry proteins of Bacillus thuringiensis, would alter the rhizosphere bacterial community. Fine roots of field cultivated Bt-maize and three conventional maize varieties were analyzed together with coarse roots of the Bt-maize. A total of 547 000 sequences were obtained. Library coverage was 100% at the phylum and 99.8% at the genus rank. Although cluster analyses based on relative abundances indicated no differences at higher taxonomic ranks, genera abundances pointed to variety specific differences. Genera-based clustering depended solely on the 49 most dominant genera while the remaining 461 rare genera followed a different selection. A total of 91 genera responded significantly to the different root environments. As a benefit of pyrosequencing, 79 responsive genera were identified that might have been overlooked with conventional cloning sequencing approaches owing to their rareness. There was no indication of bacterial alterations in the rhizosphere of the Bt-maize beyond differences found between conventional varieties. B. thuringiensis-like phylotypes were present at low abundance (0.1% of Bacteria) suggesting possible occurrence of natural Cry proteins in the rhizospheres. Although some genera indicated potential phytopathogenic bacteria in the rhizosphere, their abundances were not significantly different between conventional varieties and Bt-maize. With an unprecedented sensitivity this study indicates that the rhizosphere bacterial community of a GM maize did not respond abnormally to the presence of three insecticidal proteins in the root tissue.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp

Aims: Some Cry proteins produced by the soil bacterium Bacillus thuringiensis (Bt) or by transgenic Bt plants persist in agricultural soils for an extended period of time, which may pose a hazard for nontarget soil organisms. The aims of our study were to screen for soil fungi capable of degrading the Cry1Ac toxin and to identify the mechanisms that lead to the inactivation of this protein. Methods and Results: Of the eight fungal strains screened, only one, Chrysosporium sp., was found to produce extracellular proteases capable of degrading the 66‐kDa Cry1Ac at the N‐terminal end of amino acid 125 (alanine). The proteolytic products of the Cry1Ac toxin did not exhibit any insecticidal activity against Helicoverpa armigera, in contrast to its high toxicity exhibited in the native form. Conclusions: Proteases elaborated by the Chrysosporium sp. degrade the Cry1Ac toxin in a way that it looses its insecticidal activity against H. armigera. Significance and Impact of the Study:  Chrysosporium sp., a specific soil micro‐organism capable of producing proteases that degrade the Cry1Ac toxin into inactive products under controlled conditions is being reported for the first time. Application of this observation needs to be further tested in field conditions.