Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae)

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria.

Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.     

The C-terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding

The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain-function relationships of the toxic fragment, hybrid crystal proteins based on CryIA(b) and CryIC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both dispalyed an insecticidal spectrum similar to that of the parental crystal protein from which the C-terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C-terminal part of the toxic fragment in receptor binding. These results demonstrate that the C-terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

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

Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.     

Susceptibility of dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins

Dipel-resistant and -susceptible strains of Ostrinia nubilalis (Hübner) were evaluated for larval mortality and growth inhibition when fed diets containing individual Bacillus thuringiensis protoxins. Resistance ratios for four of the protoxins in Dipel (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) were 170-, 205-, 524-, and > 640-fold, respectively, considerably higher than the 47-fold resistance to Dipel. The Dipel-resistant strain was 36-fold resistant to Cry1Ba, a protoxin not present in Dipel. Another non-Dipel protoxin, Cry1Ca, did not cause significant mortality for either resistant or susceptible larvae with doses as high as 1.0 mg/ml. In an evaluation of larval growth inhibition, resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ba was significant at concentrations of 0.054 and 0.162 microg/ml. However, growth inhibition with Cry2Aa was not significant at either dose. These data provide information on the spectrum of resistance and cross-resistance to individual Cry protoxins in this strain.     

Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

Potential of the Bacillus thuringiensis toxin reservoir for the control of Lobesia botrana (Lepidoptera: Tortricidae), a major pest of grape plants

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

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.     

Effect of Bacillus thuringiensis Cry1 toxins in insect hemolymph and their neurotoxicity in brain cells of Lymantria dispar

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 microg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 microg/0.2 g fresh wt). Cry1E and Cry1Ac (5 microg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 microg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 microg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

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

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

Molecular and insecticidal characterization of a Cry1I protein toxic to insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 microg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.     

Toxicity of Bacillus thuringiensis delta-endotoxins against bean shoot borer (Epinotia aporema Wals.) larvae, a major soybean pest in Argentina

The toxicity of seven Bacillus thuringiensis Cry protoxins was tested against neonate larvae of Epinotia aporema, a major soybean pest in Argentina and South America. The most active protoxins were Cry1Ab and Cry1Ac, with LC50 values of 0.55 and 1.39 microg/ml, respectively. Cry1Aa, Cry1Ba, Cry1Ca, and Cry9Ca protoxins were equally toxic with LC50 values about 4 microg/ml, whereas Cry1Da was not toxic. The synergistic activity of different protoxin-mixtures was also analyzed, no synergistic effect between the Cry proteins was observed, with the exception of the poorly toxic Cry1Ba/Cry1Da mixture that was slightly synergistic. The binding capacity of individual Cry1 and Cry9Ca toxins to brush border membranes of E. aporema was also determined. The non-toxic Cry1Da toxin was the only toxin unable to bind to E. aporema membranes. In addition the heterologous competition experiments showed that Cry1Ab and Cry1Ac toxins share a common binding site. Based on these data, we propose that Cry1Ab and Cry1Ac toxins could be used in the biological control of E. aporema.     

Expression of the p20 Gene from Bacillus thuringiensis H-14 Increases Cry11A Toxin Production and Enhances Mosquito-Larvicidal Activity in Recombinant Gram-Negative Bacteria

Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10⁴ to 10⁵ cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His₆) on a nickel chelating column, whereas Cry11A produced without the P20(His₆) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.     

Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the Insect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis

Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC₅₀ values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and Cry1Ac were marginally toxic to all strains, whereas the rest of the toxins tested (Cry1Ba, Cry2Aa, and Cry2Ab) were non toxic. Significant differences in susceptibility among strains were also found for Cry1Da, being the FRA strain 25-fold more susceptible than the HOL strain. Growth inhibition, as an additional susceptibility parameter, was determined in the FRA strain with the 9 toxins. The toxicity profile obtained differed from that observed in mortality assays. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa toxins produced a similar larval growth inhibition. Cry2Aa had a lower but clear effect on larval growth inhibition, whereas Cry1Ba and Cry2Ab did not have any effect.     

Study of the activity of a new glycopeptide antibiotic eremomycin combined with tobramycin against Staphylococci in vitro

Eremomycin is an original natural antibiotic with glycopeptide structure isolated at the Institute of New Antibiotics, the USSR Academy of Medical Sciences. Activity of eremomycin alone or in combination with tobramycin was studied with using 25 clinical strains of staphylococci. 56 and 88 per cent of the strains were respectively resistant to gentamicin and kanamycin, two aminoglycoside antibiotics. All the staphylococcal strains were sensitive to eremomycin in concentrations of 0.12 to 1 microgram/ml. The MIC of tobramycin for 10 (40 per cent) sensitive strains ranged within 0.25-2 micrograms/ml. For 60 per cent of the strains the MIC was equal to or higher than 16 micrograms/ml. When eremomycin was used in combination with tobramycin the antibacterial effect with respect to 17 strains (68 per cent) increased. In 32 per cent of the strains the effect was synergistic and in 36 per cent of the strains it was additive. Indifference and antagonism were detected with respect to 7 (28 per cent) and 1 (4% per cent) strains respectively. No significant difference was shown in manifestation of the synergistic-additive nature of eremomycin and tobramycin interaction with respect to the tobramycin sensitive and resistant strains.