Identification of a delta-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29

At least three different insecticidal crystal protein genes were shown to be expressed in Bacillus thuringiensis subsp. aizawai 7.29, a strain that is potentially active against the cotton leafworm Spodoptera littoralis Bdv. Among crude K-60 fractions (60- to 70-kilodalton [kDa] molecules) that were products of proteolysed crystals containing the active domains of the protoxin molecules, we were able to distinguish several distinct components on the basis of their antigenic relationship and their larvicidal properties. A purified fraction designated SF2 was a 61-kDa component specifically active against Pieris brassicae L. and homologous to the B. thuringiensis subsp. berliner 1715 plasmid-encoded crystal protein. A second fraction designated SF1 was composed of 63- and 65-kDa polypeptides and was specifically active against S. littoralis. The SF1 fraction and particularly the 65-kDa component were not antigenically related to the 61-kDa component. The purified fractions were compared with the products of three different crystal protein genes we previously cloned from total DNA of B. thuringiensis subsp. aizawai, among them a new type of crystal protein gene encoding a protein that is specifically active against S. littoralis and other insects of the Noctuidae family. This approach led us to consider the 65-kDa component as a minimum active part of a delta-endotoxin that is encoded by this new gene. Products of the two other cloned genes can be correlated with the 61- and 63-kDa components, respectively. Thus, in B. thuringiensis subsp. aizawai 7.29, multiple delta-endotoxin genes of different structural types direct the synthesis of several delta-endotoxins with different host specificities which were identified as components of the insecticidal crystals.     

Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation

The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a cryIA(a) δ-endotoxin gene, were transferred into several Bacillus thuringiensis subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 × 10^-8 to 2 × 10^-6 transductant per CFU, depending on the strain and on the plasmid. In Cry^- and Cry⁺ native recipients, the introduction of the cryIA(a) gene resulted in the formation of large bipyramidal crystals that were active against the insect Plutella xylostella (order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into B. thuringiensis subsp. entomocidus, aizawai, or israelensis by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene cryIIIA, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various B. thuringiensis recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned δ-endotoxin genes in the different recipients.     

Expression of 135-kDa Insecticidal Protein Gene from Bacillus thuringiensis in the Yeast Saccharomyces cerevisiae

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CrylA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5′-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.     

Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.     

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of δ-endotoxin on M medium, which contained 330 μg of potassium per ml, but not on ST and ST-a media, each of which contained only 11 μg of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-β-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH₂PO₄ (3.7 mM) led to the formation of crystalline δ-endotoxin. The replacement of KH₂PO₄ with equimolar amounts of KCl, KNO₃, and potassium acetate or an equivalent amount of K₂SO₄ had a similar effect, whereas the addition of an equimolar amount of NaH₂PO₄ or NH₄H₂PO₄ did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced δ-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-β-hydroxybutyric acid granules on the media containing ≤1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of δ-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-β-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and δ-endotoxin formation but also in sporulation.     

Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

Thirty-five strains of the entomopathogenic bacterium Bacillus thuringiensisactive on Spodoptera exigua, were characterized by means of serological identification and determination of crygene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exiguaand tested against two other species of the same genus: S. littoralisand S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstakiwere the most frequent within S. exigua-active strains and that serovar aizawaihad the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in crygenes as determined by PCR suggests a non random distribution of some crygenes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodopteraspecies, were very common within S. exigua-active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralisor S. frugiperda; and some strains lacking all of the Spodoptera-active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodopteraspecies. Since strains sharing the same crygenes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensisstrains.     

Immunological properties of entomocidal protein of Bacillus thuringiensis and its insecticidal activity

The immunological properties of the proteinaceous component of the parasporal crystal (δ-endotoxin) of Bacillus thuringiensis var. kurstaki were analyzed by rocket immunoelectrophoresis. Two antisera, one against the k-l-type crystal containing two components, and the other against the k-73-type crystal containing one component, were made in rabbits. The antigens consisting of purified and dissociated crystals were run in electrophoresis with these two antisera. The ratio between the two peak heights of precipitin lines, which were formed by the dissociated crystal of one B. thuringiensis isolate in two antisera, was compared with the ratios of other isolates under identical conditions. The difference in the ratio reflected a difference in the structure of the crystal component and correlated closely with the insecticidal activity spectrum. This method can be used to evaluate a newly isolated B. thuringiensis, and it can further differentiate the isolates which have been classified as one serotype.     

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

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

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Bacillus thuringiensis subsp. Aizawai δ-endotoxin inhibits the k+/amino acid cotransporters of lepidopteran larval midgut

1. The δ-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K⁺-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae.2. The decrease of uptake in the absence of a K⁺-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin.3. The toxin acts as a non competitive inhibitor of the K⁺/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system.     

Characterization of a novel mosquitocidal strain of Bacillus thuringiensis serovar aizawai which harbors a rolling-circle replication plasmid,pBt1–3

Bacillus thuringiensis 1–3, isolated from a Korean soil sample, was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test, and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with specific cry gene primers showed that B. thuringiensis 1–3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A genes, differing from that of serovar of aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D genes. In contrast to the reference strain, B. thuringiensis aizawai showed insecticidal activity against Plutella xylostella larvae, the B. thuringiensis 1–3 showed insecticidal activity against not only P. xylostella, but also Aedes aegypti, owing to its Cry2A crystal protein. In this study, we modified the plasmid capture system (PCS) through in vitro transposition to clone small cryptic plasmids from B. thuringiensis 1–3. Fifty-three clones were acquired, and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified into four groups, showing similarities with four known B. thuringiensis plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of the pGI3-like clones, pBt1–3, was fully sequenced, and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1–3 showed high similarity with pGI3, which is of the rolling-circle replication (RCR) group VI family.     

Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. aizawai toward Lymantria dispar larvae

Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl -D-N,N- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae (Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.     

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella)

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.     

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F₄ to F₈) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F₉ found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F₉. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F₉ to F₁₃) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F₁₅). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F₂ progeny from a backcross of F₁ progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.     

Asporogenous Bacillus thuringiensis Mutant Producing High Yields of δ-Endotoxin

An asporogenous Bacillus thuringiensis subsp. kurstaki strain IK mutant, strain 290-1, which produced high yields of δ-endotoxin, was obtained by ethyl methane sulfonate treatment of a spore suspension. The mutant strain produced about the same amount of δ-endotoxin as that produced by the parent strain, but 10¹⁰ to 10¹¹ cells did not form any detectable dormant spores.