Oral Immune Priming Treatment Alters Microbiome Composition in the Red Flour Beetle Tribolium castaneum

Abstract

It is now well-established that the microbiome is relevant for many of an organism's properties and that its composition reacts dynamically to various conditions. The microbiome interacts with host immunity and can play important roles in the defenses against pathogens. In invertebrates, immune priming, that is, improved survival upon secondary exposure to a previously encountered pathogen, can be dependent upon the presence of the gut microbiome. However, it is currently unknown whether the microbiome changes upon priming treatment. We here addressed this question in a well-established model for immune priming, the red flour beetle Tribolium castaneum exposed to the entomopathogenic bacterium Bacillus thuringiensis (Bt). After priming treatments, the microbiota composition of beetle larvae was assessed by deep sequencing of the V1-V2 region of the bacterial 16S rRNA gene. We compared the effect of two established routes of priming treatments in this system: injection priming with heat-killed Bt and oral priming via ingestion of filtered sterilized bacterial spore culture supernatants. For oral priming, we used several strains of Bt known to vary in their ability to induce priming. Our study revealed changes in microbiome composition following the oral priming treatment with two different strains of Bt, only one of which (Bt tenebrionis, Btt) is known to lead to improved survival. In contrast, injection priming treatment with the same bacterial strain did not result in microbiome changes. Combined with the previous results indicating that oral priming with Btt depends on the larval microbiome, this suggests that certain members of the microbiome could be involved in forming an oral priming response in the red flour beetle.

Keywords: Bacillus; Tribolium castaneum; bacteria; infection; injection priming; insect immunity; microbiome; oral immune priming.

FIGURE 1

Overview of the experimental design and sample preparation for sequencing.

FIGURE 2

Relative microbiome composition of red flour beetle larvae based on bacterial 16S rRNA gene profiling after seven different priming treatments in two time points (A: 24 h after priming, B: 4 days after priming). The y-axis indicates relative abundances (sums to 1 for each treatment) of all the ASVs detected. Each treatment corresponds to 12 replicates of 10 pooled larvae each. Only top 10 genus have been represented in the figure with all other genus being aggregated as Others. Not_Assigned represents all ASVs for which genus could not be assigned. For the absolute microbiome abundance, see Supplementary Figure 4.

FIGURE 3

Alpha biodiversity measures in relation to time points and treatment regimes. Each dot represents one replicate of 10 pooled larvae. The letters indicate the differences between the treatments after post hoc and p-value adjustment. (A) Observed species richness measure of experimental treatments 24 h and 4 days after priming shows no difference between the regimes (24 h: ANOVA, F = 0.95, Df = 6, p = 0.47; 4 days: ANOVA, F = 0.90, Df = 6, p = 0.50). (B) Shannon index measures of experimental treatments 24 h after priming shows a higher diversity in oral Btt and oral Bt tolworthi treatments (ANOVA Df = 6, F = 5.92, p = 0.003) and 4 days after priming showing no difference between the treatments (ANOVA, F = 2.30, Df = 6, p = 0.060).

FIGURE 4

Principal—coordinate (PCoA) plot of the first time point (24 h after priming) based on Bray—Curtis dissimilarity distances. Priming with Btt and Bt tolworthi have an impact on the microbiome composition. Ellipses are drawn around samples belonging to the same priming regime and there are six replicates of each of the regimes. Permanova: F = 2.179, R² = 0.284, p < 0.001. Perdmadisp: F = 2.170, p = 0.071.

FIGURE 5

Linear discriminant analysis effect size plot. Significant features are ranked in decreasing order by their LDA scores (x-axis). The heatmap to the right of the plot indicates whether the taxa are higher (red) or lower (blue) in each treatment. FDR < 0.05, LDA score >2. Using BLAST all ASVs were identified as Bacillus.

FIGURE 6

T. castaneum survival upon different priming treatments. (A) There is no increase in survival of 19-day-old beetle larvae orally infected with Bt tolworthi spores (5 × 10⁹ mL^–1) when previously primed with Btt or Bt tolworthi supernatants [Cox proportional hazards (coxph): X² = 4.74, Df = 1.96, p = 0.09]. (B) Survival of 19-day-old beetle larvae orally infected with Btt spores (5 × 10⁹ mL^–1) is increased when previously primed with Btt supernatant [Cox proportional hazards (coxph): X² = 5.9, Df = 2, p = 0.05]. 5 days prior to the spore infections, larvae were exposed to priming diet mixture with flour and either sterile supernatants of Btt, Bt tolworthi or control Bt medium.