Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

Abstract

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

Keywords: bioinsecticide; immunity; insect biocontrol; insect–pathogen interactions; pore-forming toxins.

Fig. 1.

Cellular and humoral immune response by S. littoralis larvae as affected by RNAi-mediated silencing of the immune gene 102 Sl. (A) The transcript level of 102 Sl was significantly down-regulated in S. littoralis larvae by oral administration of 102 Sl dsRNA (P < 0.0001, t = 18.98, df = 34, n = 18). (B) RNAi-mediated silencing of 102 Sl significantly reduced the number of nodules present in the hemolymph of S. littoralis larvae that received an injection of E. coli cells (P < 0.0001, t = 25.994, df = 27, n = 28). (C) In contrast, phagocytosis of fluorescein-conjugated E. coli cells was not influenced by gene silencing. (D) The transcript level of genes encoding the humoral effectors considered was significantly enhanced by the immune challenge (n = 8 for each sampling point; attacin 1: F_{3, 66} = 198.13, P < 0.0001; gloverin: F_{3, 66} = 39.58, P < 0.0001; lysozyme1a: F_{3, 66} = 95.60, P < 0.0001), but was not influenced by gene silencing. The values reported are the mean ± SE. T₀ is the time of injection, T₁ is 18 h after injection. Different letters denote significant differences between treatments compared within each gene considered.

Fig. 2.

Midgut morphology of experimental S. littoralis larvae. (A and B) Semithin cross-sections of the midgut epithelium from control larvae treated with GFP dsRNA (A) or 102 Sl dsRNA (B). The epithelial monolayer is intact, showing regular columnar cells (cc) and goblet cells (gc) and evident PM (pm; arrowhead), delimiting the ectoperitrophic space (es). (C and D) Semithin cross-sections of the midgut epithelium from control larvae treated with GFP dsRNA (C) or 102 Sl dsRNA (D) and fed with Cry1Ca. The toxin induces severe alterations in both samples and disrupts the integrity of the intestinal barrier. Alterations of cell morphology are visible. The disaggregation of the PM, associated with feeding cessation, is evident (see the text for details), and the midgut lumen (l) does not show the presence of a delimited ectoperitrophic space. (E–G) Transmission electron microscopy analysis of the midgut epithelium (e) explanted from experimental larvae treated with Cry1Ca shows signs of cellular alteration (E–G) and the presence of bacterial cells close to the apical brush border of the midgut (E), with some of them located in the disrupted areas of the lining epithelium (arrowhead; F) and in the hemocoel (arrowhead), where muscle fibers (m) are evident (G). (Scale bars: 50 μm in A–D, 5 μm in E–G.)

Fig. 3.

Relative quantification of bacterial load by qRT-PCR and nodulation response. (A and B) Fold-change over time of bacterial load in S. littoralis larvae, as affected by different doses (µg/cm²) of Cry1Ca, in controls exposed to GFP dsRNA (A) and in immunosuppressed individuals that received a 102 Sl dsRNA treatment (B). The bacterial load resulted significantly influenced by all experimental factors only in the hemolymph (H) environment (dsRNA: F_{1, 176} = 22.65, P < 0.0001; Cry1Ca: F_{3, 176} = 45.65, P < 0.0001; time: F_{3, 176} = 310.36, P < 0.0001), whereas no significant changes were observed in the midgut (M). (C and D) The concurrent nodulation response in controls exposed to GFP dsRNA (C) and in immunosuppressed individuals who received 102 Sl dsRNA treatment (D) clearly evidenced an inverse correlation between bacterial growth and nodulation, which was significantly influenced by both toxin exposure (F_{2, 81} = 16.94, P < 0.0001) and gene silencing (F_{1, 81} = 30.63, P < 0.0001).

Fig. 4.

Incidence of the major bacterial taxonomic groups detected by pyrosequencing. Relative abundance of identified microbial taxa in the midgut content and hemolymph samples collected from S. littoralis larvae treated with 102 Sl dsRNA or GFP dsRNA, and exposed to various doses (µg/cm²) of Cry1Ca. Sample IDs are listed in SI Appendix, Table S4.