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. 2024 Feb 13;15:1341. doi: 10.1038/s41467-024-45532-4

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© The Author(s) 2024, corrected publication 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A CFU/fly comparing conventionally raised flies to those raised on antibiotic media. Antibiotic treatment is effective in reducing the fly bacterial load. B Germ-free flies do not show PGN-induced PER suppression. PER index of w- Germ-free (GF) and conventionally raised (CR) flies to control solutions of sucrose 1 mM and sucrose 1 mM + caffeine 10 mM and to sucrose 1 mM + PGN from E. coli K12 at 200 µg/mL. The data showed for CR flies are the same as in Fig. 1A. For (B), PER index is calculated as the percentage of flies tested that responded with a PER to the stimulation ± 95% CI. The number of tested flies (n) is indicated on top of each bar. For each condition, at least 3 groups with a minimum of 10 flies per group were used. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001 two-sided Fisher Exact Test. C–E Real-time calcium imaging using the calcium indicator GCaMP6s to reflect the in vivo neuronal activity of Gr66a-/ppk23+ neurons (Gr66a^LexA; LexAopGal80; ppk23^Gal4/UASGCaMP6s) in adult brains of flies whose proboscis has been stimulated with PGN. The expression of LexAop-Gal80 antagonizes the activity of Gal4, thus preventing the expression of GCaMP6S in Gr66a + /ppk23+ neurons. C Representative images showing the GCaMP6s intensity before and after addition of either the control water or the peptidoglycan. D Averaged ± SEM time course of the GCaMP6s intensity variations (ΔF/F0 %) for Gr66a-/ppk23+ neurons. The addition of water (n = 8) or peptidoglycan (n = 8) at a specific time is indicated by the arrow. E Averaged fluorescence intensity of positive peaks ± SEM of germ-free flies in response to Water (n = 8), Caffeine 10 mM (n = 8), Sodium chloride 250 mM (n = 8) and peptidoglycan from E. coli K12 at 200 µg/mL (n = 8). In (E), ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, non-parametric t-test, two-tailed Mann-Whitney test. Further details including raw data and exact p-values can be found in the source data file.