Gut microbiome and host TOR pathway interact to regulate predator-induced aversive memory in Drosophila melanogaster

Abstract

The gut microbiome has emerged as a key factor influencing a wide range of host physiological processes and behaviors, though the mechanisms behind these effects remain only partially understood. In this study, we explored the role of the gut microbiome in memory regulation using a parasitoid wasp-induced oviposition depression paradigm in Drosophila melanogaster. Our findings show that flies with depleted gut microbiota, either through axenic culture or antibiotic treatment, exhibited significant memory impairments. However, reintroducing the commensal bacterium Lactobacillus plantarum alone was sufficient to restore memory, while coinoculation with Acetobacter pomorum further enhanced memory performance. Hemolymph metabolomic analyses revealed reduced amino acid levels in antibiotic-treated flies, which were linked to impaired Drosophila target of rapamycin (dTOR) signaling. Additionally, genetic manipulation of dTOR or dietary supplementation with branched-chain amino acids either mimicked or rescued the memory deficits caused by antibiotic treatments. These results suggest that the gut microbiome is essential for regulating memory function by maintaining amino acid homeostasis and proper dTOR signaling, with profound implications for advancing knowledge of cognitive regulation.

Keywords: TOR; gut microbiome; memory.

Fig. 1.

Wasp exposure-induced acute and chronic egg-laying depression as a behavioral output of learning and memory. (A) The experimental diagram illustrates the procedure of wasp exposure and the oviposition assay of flies. Newly eclosed flies were kept at a density of 25 females and 5 males in a regular vial containing fly food. On day 5, flies were either exposed or unexposed to 15 female L. clavipes (LcNet) wasps for 24 h, and the number of eggs laid by the flies was counted daily. (B–J) The number of eggs laid by female flies of various genotypes with or without wasp exposure: w¹¹¹⁸ (B), NorpA³⁶ (C), Orco¹ (D), MB247-GAL4>UAS-TNT (E), Dnc¹ (F), Rut²⁰⁸⁰ (G), Amn¹ (H), Elav-GAL4>UAS-Radish-RNAi (I), MB247-GAL4>UAS-CrebB-RNAi (J). (K, L) The experimental diagram illustrates the procedure of wasp exposure and the oviposition assay of w¹¹¹⁸ flies with vial and bottle swaps from day 6 to day 10; the number of eggs laid by the flies was counted daily. (M) The number of eggs laid by female MB247-GAL4>UAS-Orb-RNAi flies with or without wasp exposure. (N, O) The experimental diagram illustrates the procedure of wasp exposure and the oviposition assay of w¹¹¹⁸ flies with regular vial and green vial swaps from day 6 to day 10; the number of eggs laid by the flies was counted daily. Data are presented as the mean ± SEM (n = 3-16 vials for each group). Statistically significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, determined by two-way ANOVA with Bonferroni post hoc tests.

Fig. 2.

Wasp-induced memory requires gut microbiota. The number of eggs laid by antibiotics (ABX)-treated (A) and axenic (B–H) w¹¹¹⁸ female flies with or without exposure to LcNet wasps for 24 h on day 5. (C and D) Axenic flies received GMT from gut homogenates of control w¹¹¹⁸ female flies, with or without filtration using a 0.2 µm filter to remove gut bacteria (filtered-GMT). (E–H) Axenic flies were inoculated with vehicle (PBS), Lp, and/or Ap beginning from day 1 of the adult stage. Data are presented as the mean ± SEM (n = 4-20 vials for each group). Statistically significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, determined by two-way ANOVA with Bonferroni post hoc tests.

Fig. 3.

Gut microbiota depletion induces metabolomic changes in Drosophila. Hemolymph samples of w¹¹¹⁸ flies were collected on day 4 with vehicle or ABX treatments during the behavioral paradigm. Metabolites were detected using UHPLC-MS/MS and analyzed with MetaboAnalyst 5.0. (A and B) PCA and volcano plots of vehicle-treated and ABX-treated samples. (C) Pie chart classification of 70 significantly altered metabolites comparing vehicle-treated and ABX-treated groups in (B). (D) KEGG pathway analysis of these 70 metabolites reveals significant alterations in amino acid metabolism in ABX-treated hemolymph samples. (E and F) Levels of specific ABX, EAAs, NEAAs, and CEAAs detected in UHPLC-MS/MS experiments. Data are presented as the mean ± SEM (n = 5 samples for each group). Statistically significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, determined by multiple unpaired t tests.

Fig. 4.

Essential BCAAs supplementation rescues ABX-induced memory impairments. The number of eggs laid by ABX-treated w¹¹¹⁸ female flies receiving a mixture of EAAs (A) and NEAAs (B) or individual amino acids, including leucine (Leu, C), isoleucine (Ile, D), valine (Val, E), arginine (Arg, F), histidine (His, G), lysine (Lys, H), phenylalanine (Phe, I), glutamine (Gln, J), and glutamate (Glu, K), with or without exposure to LcNet wasps for 24 h on day 5. Data are presented as the mean ± SEM (n = 4-20 vials for each group). Statistically significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, determined by two-way ANOVA with Bonferroni post hoc tests.

Fig. 5.

Gut microbiota regulate dTOR signaling to influence memory formation. (A) qPCR analysis comparing mRNA expression levels of specific dTOR target genes. (B and C) Representative Western blots and quantitative analyses showing that phosphorylated S6K levels are significantly down-regulated in ABX-treated samples compared to vehicle-treated samples. (D–I) The number of eggs laid by female flies of various genotypes with or without LcNet wasp exposure for 24 h on day 5: dTor^DeltaP/+ (D), Mef2-GAL4>UAS-dTor^TED (E), Tk-GAL4>UAS-dTor^TED (F), Matα4-GAL4>UAS-dTor^TED (G), Elav-GAL4>UAS-dTor^TED (H), and dTor^DeltaP/+; Elav-GAL4>UAS-dTor (I). (J–O) The number of eggs laid by ABX-treated female flies of various genotypes with or without LcNet wasp exposure for 24 h on day 5: Da-GAL4>tub-GAL80ts;UAS-dTor (J and M), Elav-GAL4>tub-GAL80ts;UAS-dTor (K and N), and MB247-GAL4>tub-GAL80ts;UAS-dTor (L and O). The temperature was maintained at 25 °C or adjusted to 28.5 °C from day 5 to day 10, as indicated. Data are presented as the mean ± SEM (n = 3-10 vials/samples for each group). Statistically significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, determined by unpaired t tests or two-way ANOVA with Bonferroni post hoc tests.