Immune system stimulation by the native gut microbiota of honey bees

Abstract

Gut microbial communities can greatly affect host health by modulating the host's immune system. For many important insects, however, the relationship between the gut microbiota and immune function remains poorly understood. Here, we test whether the gut microbial symbionts of the honey bee can induce expression of antimicrobial peptides (AMPs), a crucial component of insect innate immunity. We find that bees up-regulate gene expression of the AMPs apidaecin and hymenoptaecin in gut tissue when the microbiota is present. Using targeted proteomics, we detected apidaecin in both the gut lumen and the haemolymph; higher apidaecin concentrations were found in bees harbouring the normal gut microbiota than in bees lacking gut microbiota. In in vitro assays, cultured strains of the microbiota showed variable susceptibility to honey bee AMPs, although many seem to possess elevated resistance compared to Escherichia coli. In some trials, colonization by normal gut symbionts resulted in improved survivorship following injection with E. coli. Our results show that the native, non-pathogenic gut flora induces immune responses in the bee host. Such responses might be a host mechanism to regulate the microbiota, and could potentially benefit host health by priming the immune system against future pathogenic infections.

Keywords: antimicrobial peptides; apidaecin; innate immunity; symbiosis.

Figure 1.

The honey bee gut microbiota up-regulates host expression of AMPs. Two experiments are shown: (a) bees inoculated with guts of hive bees (treatment) or guts of newly emerged bees lacking microbiota (control); (b) bees inoculated with cultured S. alvi strain wkB2 (treatment), or sterile sucrose solution (control). Relative expression values indicate fold-change compared to the mean value of control.

Figure 2.

The gut microbiota alters levels of immunity-related proteins in the gut and in the haemolymph. Relative abundances are reported as peptide spectrum matches per microlitre of sample (gut fluid or haemolymph). Solid lines denote means; dashed line indicates the detection threshold.

Figure 3.

Survival rates of uninoculated and inoculated bees after E. coli infection. Bees were each injected with 1 µl of E. coli culture with 10³ cells (a,b), 10⁴ cells (c,d) or 10⁴ cells after 6 h of starvation (e). Bees inoculated with S. alvi wkB2 or G. apicola wkB7 exhibited higher survival rates after infection in two of five trials; significant differences from uninoculated treatment are denoted by asterisks. (a) wkB2, p < 0.0001; wkB7, p = 0.001; (b) wkB7, p = 0.0148; (c–e) results were not significant (log-rank test).

Figure 4.

Escherichia coli CFUs per microlitre of haemolymph in uninoculated bees and bees inoculated with cultured S. alvi, G. apicola or crushed guts at 2 and 6 h after infection with 10⁵ E. coli cells. (a) Trial 1. Inoculated bees cleared more bacteria after E. coli infection, as compared to uninoculated bees (unpaired t-test). (b) Trial 2. At 6 h after E. coli infection, bees inoculated with S. alvi wkB2 and G. apicola wkB7 cleared more bacteria from haemolymph than uninoculated bees (unpaired t-test). (c) Trial 3. There was no difference in the amount of bacteria remaining between the control and treatment groups (unpaired t-test).