Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

Abstract

Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity.

Keywords: Bt; experimental evolution; immune response; insect; microevolution; resistance.

Figure 1.

Basal (uninfected) and Bt induced (48 pi) expression of defense genes in the midgut (A) and fat body (B) of G. mellonella larvae. Expression of antimicrobial peptide genes and other immunity /stress-management genes in the fat body and midgut of resistant (R) and susceptible (S) fourth instar larvae. Expression was assessed under basal (uninfected) conditions and Bt-treated (infected) conditions 48 h post-infection. The y-axis represents basal expression in uninfected/infected R larvae as a fold change relative to S uninfected/infected larvae. Na = not assayed in midgut tissue; = *p < 0.05; = **p < 0.01; = ***p < 0.001 significant change in fold expression compared with S larvae; #-p < 0.05, ## = p < 0.01 show significant changes in expression of genes grouped in functional clusters in R vs S insects under Bt infection compared with uninfected R vs S. Data presented as mean ±SE and analyzed by one-way ANOVA (Kruskall-Wallis with Dunn's post test). Tables (cluster analysis) present trends in expression of defense genes grouped in clusters (arrow indicates significant upregulation, fold change cutoff ≥2 .0). Additional information is presented in Table S1.

Figure 2.

Lysozyme activity in infected and uninfected R and S line larvae. Lysozyme-like activity in midgut of fourth instar larvae from both susceptible and resistant wax moth lines 48 h following ingestion with Bt (data presented as mean +/−SEM; *P < 0.05, **P < 0.01, compared with uninfected larvae from the same line).

Figure 3.

Midgut receptors of uninfected R and S line larvae Aminopeptidase-N (AMN) (A) and alkaline phosphatase (ALP) (B) activity in the midgut of fourth instar uninfected larvae from both the susceptible and resistant lines (*** p < 0.001 compared with susceptible).

Figure 4.

Gut biota profiles in Bt infected and uninfected R and S line larvae. Profile of the bacterial community in midguts from fourth instar larvae from both resistant and susceptible lines on the second day post Bt infection. Values are averaged across 4 independent control (uninfected) and 4 infected samples of each line. (A) Bacteria classified by phylum and (B) Comparison of community, classified by class, from infected and uninfected R and S line larvae (p < 0.01, p < 0.001 compared with infected insects from the corresponding line).

Figure 5.

Richness and diversity of bacterial communities in infected and uninfected R and S lines (A) Chao community quantitative index reflecting richness (i.e. different bacterial phylotypes) in a dataset. (B) Shannon index reflecting diversity of bacterial communities for resistant and susceptible lines following infection with Bt (**p < 0.01, compared with other variants; *p < 0.05 compared with same non-infected line). This index quantifies how evenly the basic entities (such as phylotypes) are distributed. To prevent bias due to sampling depth, all samples were first rarefied (randomly standardized) to 3 700 sequences per sample.

Figure 6.

Increased fecundity: a positive trade-off in wax moth resistant to Bt Life-history traits in uninfected susceptible and resistant lines of 20th generation wax moth. (A) Pupal weights and (B) adult fecundity as measured by mean egg production over 5 d per female (*p < 0.05, *** p < 0.001 compared with susceptible line).