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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. 6 — A Graphical representation of the protocol to contaminate Germ-free larvae at different stage of the larvae development. Two ovipositions of yw^- flies on standard medium and w- flies on antibiotic-enriched medium were started simultaneously. After 4 h the embryos of the w^- flies are sterilized using bleach and transferred onto antibiotic-enriched media. Upon reaching the desired stage of development the w^- germ-free larvae are transferred to the same tube in which the yw^- larvae are growing. Once the adult stage is reached, flies are separated according to body color and their PER response assayed. B Co-habitation with bacteria only during the larval stage is sufficient to trigger PGN-induced PER suppression. PER index of w- flies, shifted from germ-free to conventional raising conditions (and the reverse) upon pupation, to control solutions of sucrose 1 mM + PGN from E. coli K12 at 200 µg/mL. The data showed for conventionally raised (CR) and germ-free (GF) flies are the same as in Figs. 1A and 5B. C PER index of w- Germ-free animals, shifted to conventional raising conditions at different stages of their larval development (as in Fig. 6A), to solutions of sucrose 1 mM + PGN from E. coli K12 at 200 µg/mL. The graphic represents how many days after the egg laying (AEL) larvae were transferred to yw^- used media, and their response to stimulation with PGN. D PER index of w- germ-free animals, transferred for short periods of time of their development under conventional raising conditions, to solutions of sucrose 1 mM + PGN from E. coli K12 at 200 µg/mL. The graph represents the timing of the shift from one condition to another. For (B–D), 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 < 0.001, **** indicates p < 0.0001 two-sided Fisher Exact Test. Further details including raw data and exact p-values can be found in the source data file.