1. Neuroscience

Cholesterol taste avoidance in Drosophila melanogaster

  1. Roshani Nhuchhen Pradhan
  2. Craig Montell
  3. Youngseok Lee  Is a corresponding author
  1. Department of Bio and Fermentation Convergence Technology, Kookmin University, Republic of Korea
  2. Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California, Santa Barbara, United States
https://doi.org/10.7554/eLife.106256.2
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  1. Roshani Nhuchhen Pradhan
  2. Craig Montell
  3. Youngseok Lee
(2025)
Cholesterol taste avoidance in Drosophila melanogaster
eLife 14:RP106256.
https://doi.org/10.7554/eLife.106256.2
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5 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
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The neuronal response of the adult flies to cholesterol.

(A) Schematic diagram of the fly labellum. (B) Average frequencies of action potential generated from S7, I8, and L6 sensilla upon application of different concentrations of cholesterol (CHL; n=10–12). (C) Representative sample traces of S7, I8, and L6 from (B). (D) Electrophysiological responses of control flies produced from all labellum sensilla in response to 0.1% cholesterol (n=10–12). (E) Electrophysiological analysis of S7 sensilla in response to 0.1% cholesterol using flies in which different GRNs were inactivated by the inwardly rectifying potassium channel Kir2.1 (n=10–12). (F) Representative sample traces of the S7 sensilla from (E). All error bars represent SEMs. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group (**p<0.01).

Figure 1—figure supplement 1
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Electrophysiological responses using different doses of methyl-β-cyclodextrin (MβCD).

(A) Dose-dependent neuronal responses of w1118 adult flies to MβCD from S7, I8, and L6 sensilla (n=10). (B) Representative sample traces corresponding to the data in (A). Error bars represent standard errors of the means (SEMs). Statistical analysis was performed using single-factor ANOVA with Scheffe’s post hoc analysis to compare multiple datasets.

Figure 2 with 1 supplement
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Ionotropic receptors (IRs) are responsible for sensing cholesterol.

(A) Tip recordings using 0.1% cholesterol to analyze the responses of S7 sensilla from control flies and from 32 Ir mutants (n=10–16). (B) Tip recordings using 0.1% cholesterol to analyze responses of S7 sensilla from Ir7g2, Ir25a Df/Ir25a2, Ir51b2, Ir56d2, and Ir76b2 (n=10–16). (C) Tip recordings using 0.1% cholesterol to analyze responses of S7 sensilla after RNAi knockdown of the following genes using either the Gr33a-GAL4 or ppk23-GAL4: Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b. (D) Representative sample traces of (F) for control, mutants, and rescue lines using the GAL4/UAS system. (E) Heatmap representing the dose responses (spikes/sec) elicited by S7 sensilla from the control and the indicated mutants (Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1) (n=10–16). (F) Tip recordings performed on S7 sensilla (0.1% cholesterol) from control, Ir7g1, Ir25a2, Ir51b1, Ir56d1, Ir76b1, and from flies expressing the indicated cognate transgenes under control of either their own GAL4 or the Gr33a-GAL4 (n=10–14). All error bars represent SEMs. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Black asterisks indicate statistical significance compared to the control group. The red asterisks indicate statistical significance between the control and the rescued flies (**p<0.01).

Figure 2—figure supplement 1
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Electrophysiological analyses of S7 sensilla from mutants disrupting different bitter GRs andTRP channels in the presence of 10–1% CHL, and a subset of bitter GRNs express Ir56d .

(A) Tip recordings from S7 sensilla (using 0.1% cholesterol) from mutants disrupting broadly tuned bitter GRs (n=10). (B) Neuronal response analyses from S7 sensilla from trp mutant lines using 0.1% cholesterol (n=10). (C, D, E) Tip recording analyses of control flies and candidate Irs mutant flies (Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1) with 10–3% stigmasterol (STG) from S6, S7, and S10 sensilla (n=10–12). (F) Relative spatial distributions of the Gr66a (green; anti-GFP) and Ir56d (red; anti-DsRed) reporters in the labella of Gr66a-I-GFP, Ir56d-GAL4/UAS-DsRed flies. Images were acquired by confocal microscopy. The scale bars represent 50 µm. All error bars represent SEMs. Statistical analysis was performed using single-factor ANOVA with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group (**p<0.01).

Figure 3 with 1 supplement
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Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b are required for the perception of cholesterol.

(A) Binary food choice analysis of w1118 adult flies toward different doses of cholesterol. Sucrose (2 mM) was included on both sides (n=6). (B) Binary food choice analyses to test for sex-specific difference in the feeding responses toward 0.1% cholesterol (n=6). (C) Binary food choice assays to determine the effects of inactivating different GRN types on the responses to 0.1% cholesterol. +/-indicates the presence or absence of the transgene, respectively (n=6). (D) Binary food choice assays to test the reponses of Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 flies to 0.1% cholesterol (n=6). (E) Binary food choice assays to analyze the responses of Ir7g2, Ir25a Df, Ir51b2, Ir56d2, and Ir76b2 flies to 0.1% cholesterol (n=6). (F) Dose responses of control, Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 flies to different concentrations of cholesterol (10–5%, 10–4%, 10–3%, 10–2%, and 10–1%) via binary food choice assays (n=6). (G) Rescue of Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 defects by expressing the wild-type cDNAs under the control of the GAL4 drivers specific to each gene (Ir25a, Ir56d, and Ir76b) or Gr33a-GAL4 (n=6). All error bars represent SEMs. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Black asterisks indicate statistical significance compared to the control group. The red asterisks indicate statistical significance between the control and the rescued flies (**p<0.01).

Figure 3—figure supplement 1
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Binary food choice assays with CHL and methyl-β-cyclodextrin (MβCD).

(A) Dose-dependent binary food choice assays using control flies with 10–3%, 10–2%, and 10–1% MβCD containing 2 mM sucrose vs 2 mM sucrose only (n=6). (B) Dose-dependent binary food choice assay comparing cholesterol (CHL) vs MβCD food. Sucrose (2 mM) was employed on both sides (n=6). (C) Behavioral analysis of control flies after switching the red and blue dyes in the two food options (n=6). (D) Binary food choice assays using flies expressing UAS-RNAi lines for Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b with combined with UAS-Dicer2 and driven by the Gr33a-GAL4. (E) Binary food choice assays using flies expressing UAS-RNAi lines for Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b combined with UAS-Dicer2 and driven by the ppk23-GAL4 (n=6). (F) Binary food choice assays using control flies and orco1 mutants (n=6). (G) Evaluation of the role of olfactory organs in rejecting 0.1% cholesterol using binary food choice assays (n=6). All error bars represent SEMs. Statistical analysis was performed using single-factor ANOVA with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group (**P<0.01).

Figure 4
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Testing whether ectopic expression of Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b in L- and I-type sensilla confers cholesterol responsiveness.

(A) Schematic representation of ectopic expression of Irs in B GRNs under control of the Gr33a-GAL4. (B) Tip recordings conducted from I9 sensilla with 0.1% cholesterol using flies overexpressing UAS-Ir7g, UAS-Ir25a, UAS-Ir51b, UAS-Ir56d, and UAS-Ir76b in B GRNs under control of the Gr33a-GAL4 (n=10–16). (C) Schematic presentation of misexpression of Irs in A GRNs under control of the Gr5a-GAL4. (D) Tip recordings from L6 sensilla of the indicated flies expressing the indicated Irs under control of the Gr5a-GAL4 (n=10–16). (E) Binary food choice assays testing for attraction or aversion to 0.1% cholesterol in flies misexpressing Ir7g, Ir51b, and Ir56d in A GRNs (Gr5a-GAL4). The Irs were ectopically expressed in either an Ir56d1 or Ir7g1 mutant background (n=6). The red asterisks indicate the comparison of the combination of two UAS lines (Ir7g, Ir56d and Ir51b, Ir56d) driven by Gr5a-GAL4 with all the single UAS line including the combination of Ir7g and Ir51b. All error bars represent SEMs. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Black asterisks indicate statistical significance compared with the control (**p<0.01).

Author response image 1
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Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(Drosophila melanogaster)		Ir7a1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir7g1: y1 w* Mi{y+mDint2=MIC}Ir7gMI06687Bloomington Drosophila Stock CenterBDSC:42420
Genetic reagent (Drosophila melanogaster)Ir8a1:w[*]TI{w[+m*]
=TI}Ir8a(1);Bl(1)L(2)/CyO		Bloomington Drosophila Stock CenterBDSC:23842
Genetic reagent (Drosophila melanogaster)Ir10a1:w1118 Mi{GFPE.3xP3=ET1}Ir10aMB03273Bloomington Drosophila Stock CenterBDSC:41744
Genetic reagent (Drosophila melanogaster)Ir21a1: w1118; PBac{w+mC=PB}Ir21ac02720Bloomington Drosophila Stock CenterBDSC:10975Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Ir25a2Benton et al., 2009Provided by Dr. L. Voshall
Genetic reagent (Drosophila melanogaster)Ir47a1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir48a1: w1118; Mi{GFPE.3xP3
=ET1}Ir48aMB09217		Bloomington Drosophila Stock CenterBDSC:26453
Genetic reagent (Drosophila melanogaster)Ir48b1:w1118;Mi{GFPE.3xP3=ET1}Ir48bMB02315Bloomington Drosophila Stock CenterBDSC:23473
Genetic reagent (Drosophila melanogaster)Ir51b1:w1118;PBac{w+mC=PB}rowc00387 Ir51bc00387Bloomington Drosophila Stock CenterBDSC:10046
Genetic reagent (Drosophila melanogaster)Ir52a1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir52b1:w1118;Mi{GFPE.3xP3
=ET1}Ir52bMB02231/SM6a		Bloomington Drosophila Stock centerBDSC:25212
Genetic reagent (Drosophila melanogaster)Ir52c1:w1118; Mi{GFPE.3xP3
=ET1}Ir52cMB04402		Bloomington Drosophila Stock centerBDSC:24580
Genetic reagent (Drosophila melanogaster)Ir56a1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir56b1:w1118;Mi{GFPE.3xP3
=ET1}Ir56bMB09950		Bloomington Drosophila Stock CenterBDSC:27818
Genetic reagent (Drosophila melanogaster)Ir56d1:w[*];Ir56d1Bloomington Drosophila Stock CenterBDSC:81249
Genetic reagent (Drosophila melanogaster)Ir60b3Sang et al., 2024Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir62a1:y1w*;Mi{y+mDint2=MIC}Ir62aMI00895
Iml1MI00895/TM3, Sb1 Ser1		Bloomington Drosophila Stock CenterBDSC:32713
Genetic reagent (Drosophila melanogaster)Ir67a1: y1 w*; Mi{y+mDint2
=MIC}Ir67aMI11288		Bloomington Drosophila Stock CenterBDSC:56583
Genetic reagent (Drosophila melanogaster)Ir75d1:w1118;Mi{GFPE.3xP3
=ET1}Ir75dMB04616		Bloomington Drosophila Stock CenterBDSC:24205
Genetic reagent (Drosophila melanogaster)Ir76b1Zhang et al., 2013aProvided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Ir85a1:w1118;Mi{GFPE.3xP3=ET1}Ir85aMB04613 Pif1AMB04613Bloomington Drosophila Stock CenterBDSC:24590
Genetic reagent (Drosophila melanogaster)Ir92a1:w1118;Mi{GFPE.3xP3=ET1}Ir92aMB03705Bloomington Drosophila Stock CenterBDSC:23638
Genetic reagent (Drosophila melanogaster)Ir94a1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir94b1:w1118; Mi{GFPE.3xP3=ET1}Ir94bMB02190Bloomington Drosophila Stock CenterBDSC:23424
Genetic reagent (Drosophila melanogaster)Ir94c1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir94d1:y1w[;Mi{y+mDint2=MIC}
Ir94dMI01659CG17380MI01659		Bloomington Drosophila Stock CenterBDSC:33132
Genetic reagent (Drosophila melanogaster)Ir94f1: y1 w*; Mi{y+mDint2=MIC}Ir94fMI00928Bloomington Drosophila Stock CenterBDSC:33095
Genetic reagent (Drosophila melanogaster)Ir94g1: w1118; Mi{GFPE.3xP3=ET1}Ir94gMB07445Bloomington Drosophila Stock CenterBDSC:25551
Genetic reagent (Drosophila melanogaster)Ir94h1Rimal et al., 2019Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Ir100a1: w1118;P{w+mC=EP}Ir100aG19846 CG42233G19846Bloomington Drosophila Stock CenterBDSC:31853
Genetic reagent (Drosophila melanogaster)UAS-Ir25aLee et al., 2018Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)UAS-Ir51bDhakal et al., 2021Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)Gr33a1Moon et al., 2009Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Gr33a-GAL4Moon et al., 2009Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Gr47a1Lee et al., 2015Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)elav-GAL4;UAS-Dicer2Bloomington Drosophila Stock CenterBDSC:25750
Genetic reagent (Drosophila melanogaster)Gr39a1Bloomington Drosophila Stock CenterBDSC:10562
Genetic reagent (Drosophila melanogaster)Gr93a3Lee et al., 2009Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)UAS-Kir2.1Bloomington Drosophila Stock CenterBDSC:6596
Genetic reagent (Drosophila melanogaster)ΔGr32aMiyamoto and Amrein, 2008Provided by Dr. H. Amrein
Genetic reagent (Drosophila melanogaster)Gr66aex83Moon et al., 2006Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Gr89a1Korea Drosophila Resource CenterKDRC: (Sung et al., 2017)
Genetic reagent (Drosophila melanogaster)Ir7cGAL4McDowell et al., 2022Provided by Dr. M. Gordon
Genetic reagent (Drosophila melanogaster)Ir20a1Ganguly et al., 2017Provided by Dr. A. Dahanukar
Genetic reagent (Drosophila melanogaster)Ir25a-GAL4Benton et al., 2009Provided by Dr. L. Vosshall
Genetic reagent (Drosophila melanogaster)UAS-Ir76bMoon et al., 2006Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)Ir76b-GAL4Moon et al., 2006Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)ppk23-GAL4Thistle et al., 2012Provided by Dr. K. Scott
Genetic reagent (Drosophila melanogaster)ppk28-GAL4Cameron et al., 2010Provided by Dr. H. Amrein
Genetic reagent (Drosophila melanogaster)Gr5a-GAL4Dahanukar et al., 2001Provided by Dr. H. Amrein
Genetic reagent (Drosophila melanogaster)UAS-Kir2.1Bloomington Drosophila Stock CenterBDSC:6595
Genetic reagent (Drosophila melanogaster)Ir7g2Pradhan et al., 2024Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)UAS-Ir7gPradhan et al., 2024Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)UAS-Ir56dSánchez-Alcañiz et al., 2018Provided by Dr. R. Benton
Genetic reagent (Drosophila melanogaster)Ir56d-GAL4Korea Drosophila Resource CenterKDRC:2307
Genetic reagent (Drosophila melanogaster)Ir56d2Bloomington Drosophila Stock CenterBDSC:81250
Genetic reagent (Drosophila melanogaster)Ir51b2Dhakal et al., 2021Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)BC/CyO;Gr66a-I-GFP,UAS-dsred/TM6bWeiss et al., 2011Provided by Dr. J.R. Carlson
Genetic reagent (Drosophila melanogaster)Ir7g RNAiVienna Drosophila Resource CenterVDRC:100885
Genetic reagent (Drosophila melanogaster)Ir25a RNAiVienna Drosophila Resource CenterVDRC:106731
Genetic reagent (Drosophila melanogaster)Ir51b RNAiVienna Drosophila Resource CenterVDRC:29984
Genetic reagent (Drosophila melanogaster)Ir56d RNAiVienna Drosophila Resource CenterVDRC6112
Genetic reagent (Drosophila melanogaster)Ir76b RNAiVienna Drosophila Resource CenterVDRC8433
Genetic reagent (Drosophila melanogaster)trpA11Kwon et al., 2008Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)trpl29134Niemeyer et al., 1996Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)trpγ1Akitake et al., 2015Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)amo1Watnick et al., 2003Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)iav3621Bloomington Drosophila Stock centerBDSC:24768
Genetic reagent (Drosophila melanogaster)nan36aKim et al., 2003Provided by Dr. C. Kim
Genetic reagent (Drosophila melanogaster)trp343Tracey et al., 2003Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)pyx3Lee et al., 2005Provided by Dr. Y. Lee
Genetic reagent (Drosophila melanogaster)wtrwexKim et al., 2010Provided by Dr. C. Montell
Genetic reagent (Drosophila melanogaster)pain2Tracey et al., 2003Provided by Dr. S. Benzer
AntibodyRabbit anti-DsRed(rabbit polyclonal)TakaraCat # 632496
RRID:AB_10013483		1:1000 (1 µL)
AntibodyGoat anti-mouse Alexa Fluor 568Thermo fisher/InvitrogenCat # A11004
RRID:AB_2534072		1:200 (1 µL)
AntibodyMouse anti-GFP (mouse monoclonal)Molecular probeCat # A11120
RRID:AB_221568		1:1000 (1 µL)
AntibodyGoat anti-mouse Alexa Fluor 488Thermo Fisher/InvitrogenCat # A11029
RRID:AB_2534088		1:200 (1 µL)
Chemical compound or drugCholesterolSigma-Aldrich Co.Cat# C4951
Chemical compound or drugSucroseSigma-Aldrich Co.Cat# S9378
Chemical compound or drugTricholine citrateSigma-Aldrich Co.Cat# T0252
Chemical compound or drugStigmasterolSigma-Aldrich Co.Cat# S2424
Chemical compound or drugSulforhodamine BSigma-Aldrich Co.Cat# 230162
Chemical compound or drugBrilliant blue FCFWako Pure Chemical Industry Ltd.Cat# 027–12842
Chemical compound or drugMethyl beta cyclodextrinSigma-Aldrich Co.Cat# 332615
Chemical compound or drugParaformaldehydeElectron Microscopy SciencesCat # 157101:500
Provided by Dr. J.A. Veenstra
Chemical compound or drugGoat Serum, New Zealand originGibcoCat # 16210064
Software, algorithmOrigin Pro VersionOriginLab corporationRRID:SCR_002815https://www.originlab.com/
Software, algorithmGraphpad PrismGraphPadRRID:SCR_002798https://www.graphpd.com/
Software, algorithmAutospike 3.1 softwarehttps://www.syntech.co.za/

Additional files

Supplementary file 1

Electrophysiological analysis and binary food choice assay of MβCD, cholesterol, and stigmasterol, and immunohistochemical analysis of Ir56d-GAL4 co-localization with a bitter GRN reporter were performed.

https://cdn.elifesciences.org/articles/106256/elife-106256-supp1-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/106256/elife-106256-mdarchecklist1-v1.docx

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  1. Roshani Nhuchhen Pradhan
  2. Craig Montell
  3. Youngseok Lee
(2025)
Cholesterol taste avoidance in Drosophila melanogaster
eLife 14:RP106256.
https://doi.org/10.7554/eLife.106256.2