Honeybee gut Lactobacillus modulates host learning and memory behaviors via regulating tryptophan metabolism

Abstract

Honeybees are highly social insects with a rich behavioral repertoire and are a versatile model for neurobiological research. Their gut microbiota comprises a limited number of host-restricted bacterial phylotypes that are important for honeybee health. However, it remains unclear how specific gut members affect honeybee behaviors. Here, we find that antibiotic exposure disturbs the gut community and influences honeybee phenotypes under field conditions. Using laboratory-generated gnotobiotic bees, we show that a normal gut microbiota is required for olfactory learning and memory abilities. Brain transcriptomic profiling reveals distinct brain gene expression patterns between microbiota-free and conventional bees. Subsequent metabolomic analyses of both hemolymph and gut samples show that the microbiota mainly regulates tryptophan metabolism. Our results indicate that host-specific Lactobacillus strains promote memory behavior by transforming tryptophan to indole derivatives that activate the host aryl hydrocarbon receptor. Our findings highlight the contributions of specific gut members to honeybee neurological processes, thus providing a promising model to understand host-microbe interactions.

Fig. 1. Antibiotic treatment affects the phenotypes of honeybees under field conditions.

a Schematic of field experiments. Age-controlled bees were treated with tetracycline for 5 days (Days 7–11) in the hive and recovered for 7 days (Days 11–19). b Number of capped brood cells during the recovery stage (Days 17, 18, and 19) in three independent colonies of control and antibiotic-treated group, respectively. c The number of labeled workers recovered from three colonies of each group on Days 6, 13, and 19. Differences between antibiotic-treated bees and the control group were tested by multiple two-tailed t-test with Benjamini-Hochberg correction in (b) and (c). d Images of brood frames and dissected guts of control and antibiotic-treated groups. e Principal coordinate analysis of Bray-Curtis dissimilarity of gut community compositions of control and antibiotic-treated bees. Group differences were tested by permutational multivariate ANOVA (PERMANOVA). f Relative abundance of genera in metagenomic samples from control and antibiotic-treated groups. g Relative abundance of the four Lactobacillus Firm5 species (n = 5 bees for both groups). Differences between control and antibiotic-treated groups were tested by two-sided Mann-Whitney u test. ns, not significant. Error bars represent min and max. Source data are provided as a Source Data file.

Fig. 2. Gut microbiota alters honeybee learning and memory behaviors and the brain gene expression profile.

a Olfactory learning and memory test design. 7-day-old conventionalized (CV), tetracycline-treated (CV + tet), and microbiota-free (MF) bees were tested. Bees responded to only the nonanol odor were considered successful. b Ratio of bees successful in the memory test. Group differences among CV (n = 38), CV + tet (n = 41), and MF (n = 46) bees were tested by Chi-squared test. c A volcano plot showing the differentially regulated genes (FDR < 0.05 and |log2FoldChange | > 1, Benjamini-Hochberg FDR method). d KEGG pathways upregulated in the brains of CV bees based on the differentially expressed genes (Fisher’s exact test). Source data are provided as a Source Data file.

Fig. 3. Tryptophan metabolism pathways in the gut and hemolymph are influenced by gut microbiota.

a Sparse PLS-DA based on all metabolites detected in the hemolymph. Group differences were tested by permutational multivariate ANOVA (PERMANOVA). b The most enriched KEGG pathways down- and upregulated in the hemolymph of CV bees compared to MF group based on the differentially regulated metabolites (Fisher’s exact test). c A volcano plot showing the differentially regulated metabolites. d Key metabolites and enzymes of tryptophan metabolism via the kynurenine (Kyn) and indole pathways. Genes encoded by the host are shown in blue. Genes encoded by the gut bacteria are shown in red. Indole derivatives acting as AhR ligands are shown in orange. e Normalized concentration of tryptophan (Trp) and indole-3-acrylic acid (IA) in the hemolymph of MF and CV bees (n = 6 bees for both groups). The ratio of Kyn/Trp and kynurenic acid (KA)/Kyn was calculated (n = 6 bees for both groups). f Normalized concentration of Trp and indole-3-acetic acid (IAA), the Kyn/Trp ratio, and the KA/Kyn ratio in the midgut, ileum, and rectum of MF and CV bees (n = 6 bees for both groups). Group differences were tested by two-sided Mann-Whitney u test. Error bars represent min and max. Source data are provided as a Source Data file.

Fig. 4. Lactobacillus apis with ArAT alters tryptophan metabolism and improves honeybee memory retention.

a Graphical representation of the gene locus encoding ArAT (locus_tag: H3R21_07190). Neighbor-joining tree based on the amino acid sequences of ArAT. Sequences from honeybee Lactobacillus spp. and Bombiscardovia are clustered together with that of Lactobacillus reuteri from human gut. Nodes with high bootstrap values are marked (> 80%; 1000 replicates). b Experimental design: MF bees were fed with 50% sucrose with (MF + Trp) or without 12% tryptophan (MF). MF bees colonized with L. apis were provided with 50% sucrose with (Firm5 + Trp) or without 12% tryptophan (Firm5). Each group contains 50 bees. The learning and memory performance was tested at 7 days of age. c Ratio of bees successful in the memory test. Group differences among MF (n = 49), MF + Trp (n = 43), Firm5 (n = 49), and Firm5+Trp (n = 43) bees was tested by Chi-squared test. ns, not significant. d, e Boxplots of the normalized concentration of (d) Trp and (e) IAA in the gut of MF, MF + Trp, Firm5, and Firm5+Trp bees (n = 6 bees for all groups). f Relative expressions of the TDO gene in the gut of MF, MF + Trp, Firm5, and Firm5+Trp bees (n = 7 bees for all groups). Group differences of metabolite concentrations and gene expression levels were tested by two-sided Mann-Whitney u test. Error bars represent min and max. Source data are provided as a Source Data file.

Fig. 5. Lactobacillus apis promotes learning and memory behaviors in an AhR-dependent manner.

a Relative expressions of the AhR gene in the gut of MF, MF + Trp, Firm5, and Firm5+Trp bees (n = 7 bees for all groups). b Relative expressions of the AhR gene in the gut of Firm5+Trp bees treated with AhR antagonist (AhR^–) dissolved in DMSO (n = 7 bees for both groups). Group differences of gene expression levels were tested by two-sided Mann-Whitney u test. Error bars represent min and max. c Ratio of bees successful in the memory test. Group difference between the DMSO (n = 42) and AHR^– (n = 43) was tested by Chi-squared test. d Graphical summary of the effects of gut microbiota on honeybee behavioral phenotypes. Gut bacteria encoding ArAT produce indolic AhR ligands and suppress the host TDO in the Kyn pathway. An altered gut microbiota significantly impacts the hive phenotypes under field conditions, learning and memory abilities, and gene expression patterns in the brain. Source data are provided as a Source Data file.