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. 2006 Oct 10;103(41):15196-9.
doi: 10.1073/pnas.0604865103. Epub 2006 Sep 27.

Midgut bacteria required for Bacillus thuringiensis insecticidal activity

Nichole A Broderick  1 , Kenneth F Raffa, Jo Handelsman
Affiliations

Midgut bacteria required for Bacillus thuringiensis insecticidal activity

Nichole A Broderick et al. Proc Natl Acad Sci U S A. .

Abstract

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coli engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobacter sp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of antibiotic concentration on toxicity of B. thuringiensis to third-instar Lymantria dispar. Larvae were reared on sterile artificial diet that was unamended or amended with an antibiotic mixture containing up to 500 μg each of penicillin, gentamicin, rifampicin, and streptomycin per ml. Curves were fit by using PROC NLIN in SAS 9.1.3 (28). All are of the general form y = ae−bx. Dose 0 IU, a = 0.0862, b = 0.0108, F2,6 = 5.47, P > 0.05; dose 1.0 IU, a = 0.3125, b = 0.0608, F2,6 = 8.12, P > 0.02; dose 10.0 IU, a = 0.5351, b = 0.0474, F2,6 = 41.12, P > 0.0003.
Fig. 2.
Fig. 2.
Restoration of B. thuringiensis toxicity by an Enterobacter sp. after elimination of detectable gut flora and B. thuringiensis activity by antibiotics. “Antibiotic cocktail” L. dispar larvae were reared until the third instar on sterile artificial diet amended with 500 μg each of penicillin, gentamicin, rifampicin, and streptomycin per ml. Each bar represents the mean mortality ± SEM of 48 larvae (four replications of 12 larvae each). Values at the bottom represent the sizes of the populations of the Enterobacter sp. as detected by culturing. nd, not detected.
Fig. 3.
Fig. 3.
Growth of B. thuringiensis, Enterobacter sp. NAB3, and E. coli ECE52 in tryptic soy broth (Left) and L. dispar hemolymph (Right). The detection limit was 200 cfu/ml; samples in which no colonies were detected were assigned this value and are indicated by open symbols. Each point represents the mean cfu/ml ± SEM of three replicate cultures.
Fig. 4.
Fig. 4.
Insecticidal activity of a B. thuringiensis toxin-producing E. coli strain (ECE52) is abolished by heat-killing in the absence of normal gut microbiota. Larvae were fed live ECE52 with or without antibiotics, or they were fed heat-killed ECE52 with or without antibiotics, as indicated. Each bar represents the mean mortality ± SEM of 36 third-instar L. dispar (three replications of 12 larvae each).
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