Journal: PLOS Biology
Article Title: Uracil-driven ROS signaling and larval TrpA1-B neuron activation are required for priming the adult Drosophila gustatory response to bacteria
doi: 10.1371/journal.pbio.3004042
Figure Lengend Snippet: (A) Live L. brevis are necessary and sufficient to prime the larvae. Sterilized eggs were exposed to live or dead bacteria as well as supernatant. The resulting adults were raised on antibiotics-containing media (ATB). PER index of w − flies to control solutions of sucrose (Sucrose 1 mM) and sucrose + PGN from E. coli K12 (Sucrose 1mM + PGN200) were obtained. (B) Uracil is necessary and sufficient to prime the larvae. Sterilized eggs were exposed to the different treatments and the resulting adults raised on antibiotics-containing media (ATB). Specifically, eggs were either monoassociated with bacterial strains, or exposed to LB media or to LB media containing uracil or cytosine. Addition of uracil or Ecc 15 24h after sterilized egg deposition was tested. The resulting adults were raised on antibiotics-containing media (ATB). PER index of w − flies to control solutions of sucrose and sucrose + PGN from E. coli K12 at 200 µg/mL (PGN200) were obtained. (C) The Duox enzyme is necessary in enterocytes for the priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of GAL4 ( mex - GAL4 /+) and UAS (+/UAS-Duox_IR ) control flies as well as animals with the Duox transcript impaired via RNAi in enterocytes ( mex - GAL4 / UAS-Duox_IR ) were calculated. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (D) Graphical representation of the life periods during which flies are shifted from 18 °C to 29 °C for a stage-dependent RNAi assay. The ubiquitously expressed Tub-G80 ts , that inhibits the activity of GAL4, is temperature-sensitive: the GAL4 inhibitor is active at 18 °C (green) and inactivated at 29 °C (black), its inactivation allows the expression of any UAS . In the case of mex - GAL4 ; TubG80 ts /UAS-Duox_IR , at 18 °C the Duox transcripts will not be impaired (Duox) while it will be at 29 °C. (E) Duox activity in enterocytes is necessary during the larval life for priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of mex-GAL4; TubG80 ts /UAS-Duox_IR flies with the Duox transcript impaired all-life long, only during the larval stages or only during the adult stage were calculated. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (F) H 2 O 2 is detectable within the larvae 24 h following exposure to uracil 1 mM. Sterilized eggs were exposed to LB media or to LB media containing uracil. Twenty-four h later, the concentration of H 2 O 2 within the animals was quantified. To confirm that H 2 O 2 is the quantified product, catalase (0.01 U/mL and 0.001 U/mL) was applied to the samples from uracil-exposed larvae or overexpressed in enterocytes ( mex-GAL4/UAS-Catalase ). (G) Reducing the amount of ROS impairs the priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of UAS (+/UAS-Catalase ) control flies as well as animals with the Catalase over-expressed in enterocytes ( mex-GAL4/UAS-Catalase ) were obtained. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (H) H 2 O 2 supplementation to larvae is not sufficient to prime while vitamin C (VitC) addition during the larval life prevents it. PER index to solutions of sucrose + PGN (PGN200) of w − flies was calculated. Larvae were raised on conventional media with or without vitamin C (0.2 mg/mL) or on monoassociation media with or without H 2 O 2 (1% on eggs or 1% on eggs then 1% on 24h-larvae (2X) or 2% on eggs). All the resulting adults were raised on antibiotics-containing media. PER index of w − flies to solutions of sucrose + PGN from E. coli K12 at 200 µg/mL (PGN200) was obtained. The PER index is calculated as the percentage of flies tested that responded with a PER to the stimulation ± 95% confidence interval (CI). A PER value of 1 means that 100% of the tested flies extended their proboscis following contact with the mixture, a value of 0.2 means that 20% of the animals extended their proboscis. 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. Each independent group is represented as an open circle. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001 Fisher’s exact test. Further details can be found in the detailed lines, conditions and, statistics for the figure section. The data underlying this Figure can be found in . Created in BioRender. Kurz, L. (2026). https://core.local.biorender.dev/api/short-link/9ojribp .
Article Snippet: PGN is E. coli K12: Invivogen, catalog code #tlrl-kipgn.
Techniques: Bacteria, Control, RNAi Assay, Activity Assay, Inactivation, Expressing, Concentration Assay
