Toxic effects of spore/crystal ratios of Bacillus thuringiensis on European corn borer larvae

Bioassays to determine LC₅₀ values of spores and crystals of four varieties of Bacillus thuringiensis grown on nutrient agar plates were carried out against neonate and 6-day-old European corn borer, Ostrinia nubilalis, larvae. The four bacterial varieties were equally toxic against the neonates, but only B. thuringiensis var. kenyae, var. galleriae, and var. kurstaki were toxic to 6-day-old larvae. B. thuringiensis var. tolworthi was inactive against 6-day-old larvae. Different ratios of pure spores and crystals of the bacteria also were tested against neonate and 6-day-old larvae. Pure spores are not pathogenic to neonates or 6-day-old larvae. Pure crystals were toxic to both ages of the larvae, but a combination of spores and crystals was necessary for maximum larval mortality.     

Comparative effects of spore-crystal complexes and thermostable exotoxins of six subspecies of Bacillus thuringiensis on Ostrinia nubilalis (Lepidoptera: Pyralidae)

The relative activities of spore-crystal complexes and thermostable exotoxin produced by six subspecies of Bacillus thuringiensis were investigated using larvae of the European corn borer, Ostrinia nubilalis. Bacillus thuringiensis subsp. kenyae, subsp. galleriae, and subsp. kurstaki produced spore-cystal complexes active against the borer. Bacillus thuringiensis subsp. thuringiensis and subsp. darmstadiensis produced thermostable exotoxins active against the borer. Only one subspecies, B. thuringiensis subsp. tolworthi, produced both a spore-crystal complex and a thermostable exotoxin active against corn borer larvaer.     

Fermentation media and production of exotoxin by three varieties of Bacillus thuringiensis

The insecticidal activities of the exotoxin produced by three varieties of Bacillus thuringiensis grown in six fermentation media were determined by testing the supernatants against larvae of the house fly, Musca domestica, and the black cutworm, Agrotis ipsilon. The activities of the exotoxins from the isolates varied when they were grown in the same medium and also when they were grown in different media. When an isolate of B. thuringiensis var. thuringiensis, and of var. tolworthi were grown in proflo broth, the supernatants produced were more toxic to house fly than to black cutworm larvae, indicating the presence of more than one exotoxin. Autoclaving the supernatants for 15 and 30 min further demonstrated the presence of several exotoxins.     

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.     

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.     

Effect of exposure to soil on potency and spore viability of Bacillus thuringiensis

Ten-gram samples of a clay loam soil were inoculated with Bacillus thuringiensis var. galleriae (H-serotype V) and held at 25°C. Periodically the spores and δ endotoxin protein crystals of B. thuringiensis were extracted from soil samples. Numbers of viable spores were estimated by plate counts and pathogenicity determined by bioassay with larvae of Galleria mellonella. During 135 days, the number of viable spores fell slowly to 24% of the initial numbers, while pathogenicity fell rapidly to <1%, which suggests that the crystals were degraded far more rapidly than spores. Natural soil bacteria increased in numbers during the same period.     

Effect of incubation in natural and autoclaved soil upon potency and viability of Bacillus thuringiensis

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai H-serotype 7, strain HD137, streptomycin-resistant mutant, were added to normal and autoclaved aliquots of pH 5 soil incubated at 25°C and ?0.10 MPa water availability. Viable B. thuringiensis in soil samples were estimated by dilution-plating on a streptomycin-based medium, and combined spore and crystal insecticidal activity was bioassayed with larvae of Galleria mellonella. Populations of B. thuringiensis in both soil treatments suffered exponential rates of mortality, which were represented by segmented linear regression. Mortality was far greater in natural than autoclaved soil. Potency also fell in both soil treatments. This loss of potency was greater in natural soil, although the rates of potency loss in either soil treatment correlated poorly with the respective mortality rates of the B. thuringiensis populations, as potency losses were not exponential functions. The results suggest that the presence of indigenous microorganisms in natural soil accelerated the rate of mortality and loss of potency of B. thuringiensis.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Susceptibility of the pine processionary caterpillar Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) toward δ-endotoxins of Bacillus thuringiensis under laboratory conditions

A series of natural crystal proteins from B. thuringiensis subsp. Alesti 12–25, caucasicus, galleriae 11–67, galleriae 6–96, kenyae, and shondungensis and spore‐crystal preparations from finitimus 11–66 and from a recombinant strain of B. thuringiensis subsp. kurstaki expressing Cry 1 Ga1 only, were assessed as a toxic agent for the pine processionary caterpillar, Thaumetopoea pityocampa. Some preparations had a thoroughly investigated composition and contained Cry1Aa, Cry1Ab2, Cry1Ab7, Cry1D, Cry1F, Cry 1 Ga1, Cry9Aa, Cry26 crystal proteins, whereas crystals of B. thuringiensis subsp. caucasicus, kenyae, and shondungensis harboured predominantly unidentified toxins distant from commonly used prototypes. Bioassays were based on the simultaneous assignment of each treatment to groups of 20 full sibling first‐instar larvae, obtained from broods of a population from North‐western Italy. The toxin was applied to pine needles by the leaf dipping method and the effect was registered in both feeding inhibition and mortality. B. thuringiensis subsp. caucasicus, kenyae, galleriae 6–96, alesti, and galleriae 11–67 gave the best results in terms of both feeding inhibition and larval mortality. Broods tested in B. thuringiensis bioassays showed a substantial variation in susceptibility to the toxins, suggesting the potential development of resistance in the population.     

Hemolymph responses in Heliothis zea to inoculation with Bacillus thuringiensis or Micrococcus lysodeikticus

Direct injection into the hemolymph of Heliothis zea of either an entomopathogen (Bacillus thuringiensis subsp. kurstaki) or a nonpathogen (Micrococcus lysodeikticus) is followed by a rapid phagocytosis and extensive removal of the organisms within 2 hr. The bacteria that survive this initial clearance initiate a new round of growth that is clearly evident 6–8 hr after injection. When the infecting organism is M. lysodeikticus, a second period of clearance occurs 8–12 hr after injection and nearly complete removal (many by lysis) is evident by the 12th hr. Larvae usually survive infection with this organism. When B. thuringiensis is the infecting organism, 60–80% of the phagocytized bacteria are lysed, however, the second wave of clearance seen with M. lysodeikticus does not occur; instead, the bacteria multiply extensively and death of the larvae results 12–16 hr after injection. This death does not appear to be caused either by crystalline protein or by the β-exotoxin. Analysis of hemolymph proteins using one-dimensional polyacrylamide gel electrophoresis indicated that although some quantitative changes were observed in some experiments, in the faster moving proteins when the infecting agent was B. thuringiensis, they were not consistent enough to support the idea that hemolymph proteins were either synthesized or used up during the time larvae were responding to the infectious agent. Dramatic changes were evident when the larvae were near death. No changes were ever observed when M. lysodeikticus was used as the infecting organism. A rapid response to infection using free spores of B. thuringiensis (sickness within 2–4 hr followed by death at 6–8 hr) may indicate that the spore germinating process is accompanied by release of a highly toxic material.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

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.     

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.     

Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Genes Encoding the N-Acyl Homoserine Lactone-Degrading Enzyme Are Widespread in Many Subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.     

The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae

Comparative bacteriological and serological studies of three isolates and the reference strain of Bacillus thuringiensis subsp. darmstadiensis (serotype 10) were conducted. No difference was shown in the flagellar antigenic structure between the three isolates and the reference strain. Differences were observed in the O antigenic structures and in the following biochemical properties: lecithinase production, DNase production, arginine decarboxylase production, acid production from inulin, and malonate utilization. β-Exotoxin production was not detected in these three isolates. The reference strain produced parasporal inclusions toxic to the lepidopterous larvae but nontoxic to mosquito larvae. On the contrary, two among the three isolates, which produced spherical parasporal inclusions, were not toxic to the lepidopterous larvae but highly toxic to larvae of the mosquitoes, Culex tritaenlorhynchus, Culex molestus, and Aedes aegypti. Another isolate produced large irregular-shaped inclusions nontoxic to the insects of both orders. Accordingly, B. thuringiensis serotype 10 was divided into three groups from the viewpoint of toxicity against lepidopterous and mosquito larvae.     

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

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

Activité comparée de 22 variétés deBacillus thuringiensis sur 3 espèces deCulicidae

Résumé Le pouvoir larvicide des cultures totales de 22 variétés deBacillus thuringiensis Berliner représentant 15 sérotypes H a été testé sur larves L4 d'Aedes aegypti (L.)Culex pipiens pipiens (L.) etAnopheles stephensi (Liston). Seul le sérotype H 14, variétéisraelensis, est réellement actif, provoquant 100% de mortalité à la dilution 10⁻⁵. Avec des doses beaucoup plus fortes, 10⁻², une certaine toxicité peut être manifestée par les variétésentomocidus, galleriae etkyushuensis en ce qui concerneAe. aegypti etC. pipiens pipiens, ou par les variétésentomocidus, tolworthi, kyushuensis etaizawai, pourAn. stephensi. Cependant cette activité n'a rien de comparable avec celle de la variétéisraelensis.

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Summary We have studied the 15 H serotypes ofBacillus thuringiensis Berliner including 22 varieties. The larvicidal potency of the whole cultures of these varieties is evaluated on 4th instar larvae ofAedes aegypti (L.),Culex pipiens pipiens (L.) andAnopheles stephensi (Liston). The H-14 serotype, varietyisraelensis is the only one to show a true toxicity at 10⁻⁵ dilution on larvae of the 3 mosquito species. A low mortality at 10⁻² dilution is observed onAe. aegypti andCx. pipiens pipiens larvae withentomocidus, galleriae andkyushuensis varieties; onAn. stephensi withentomocidus, tolworthi, kyushuensis andaizawa? varieties. Nevertheless, this activity cannot be compared to the extremely high toxicity of theisraelensis variety.