DOI: 10.5061/dryad.qrfj6q5ww
title: Gustatory avoidance of fatty acids by <em>Aedes aegypti</em> depends on an arthropod-specific TRP channel

description: Mosquito-disease vectors, such as Aedes aegypti, use their sense of taste
 before deciding whether to consume a blood meal, or fly away. However, the
 molecular mechanisms controlling gustatory decisions in mosquitoes are
 largely unknown. The TRP channel, Painless1 (Pain1), is an intriguing
 candidate for participating in Ae. aegypti taste since pain1 transcripts
 are detected in gustatory receptor neurons (GRNs). The Drosophila homolog,
 painless (pain) is also expressed in GRNs, where it is required for
 sensing allyl-isothiocyanate. Here, to identify additional gustatory roles
 for pain homologs, we first focused on Drosophila pain, which is widely
 expressed in multiple GRNs classes. We demonstrated that pain mutations
 eliminated gustatory attraction to low fatty acids levels, repulsion to
 high levels, and fatty acid-induced action potentials. The attraction and
 repulsion depended on pain expression in different GRN classes. In
 contrast to Drosophila, when Aedes contacts fatty acids, they induce
 gustatory rejection only. Aedes pain1 is expressed in taste organs, and is
 required for gustatory avoidance of fatty acids, and for fatty
 acid-induced action potentials. Given that Pain homologs are found in
 insects but not vertebrates, Pain1 represents an intriguing target for
 developing repellents to diminish biting, and transmission of infectious
 agents by mosquito disease vectors.
description: Data were generated using established behavioral,
 electrophysiological, molecular, and imaging approaches
 in <em>Drosophila melanogaster</em> and <em>Aedes
 aegypti</em>, as described in detail in the associated article.
 Proboscis extension response (PER) assays were performed by stimulating
 the labella or tarsi with sucrose alone or sucrose mixed with fatty acids
 or other tastants, and scoring feeding responses. Electrophysiological
 recordings were obtained using extracellular tip-recording techniques from
 individual labellar taste sensilla following chemical stimulation, and
 action potential frequencies were quantified over defined analysis
 windows. Gene expression was assessed using RT-PCR and quantitative
 real-time PCR (RT-qPCR), with ribosomal protein genes used as internal
 controls. Confocal microscopy was used to image gustatory receptor neuron
 populations following immunohistochemistry or reporter expression, and
 scanning electron microscopy (SEM) was used to characterize labellar
 sensilla morphology. Full experimental protocols, sample sizes, and
 statistical methods are provided in the main article Methods and figure
 legends.
description: # Gustatory avoidance of fatty acids by *Aedes aegypti* depends on an
 arthropod-specific TRP channel Dataset DOI:
 [10.5061/dryad.qrfj6q5ww](https://doi.org/10.5061/dryad.qrfj6q5ww) ##
 Description of the data and file structure These data support the
 manuscript entitled “Gustatory avoidance of fatty acids by *Aedes aegypti*
 depends on an arthropod-specific TRP channel.” The dataset consists of two
 compressed folders, Raw_Data.zip and Source_Data.zip, which together
 contain the raw and processed data underlying the figures and
 supplementary figures presented in the manuscript.\ \ The deposited data
 include the raw data corresponding specifically to the images, traces, and
 gels displayed in the manuscript, as well as the full quantitative source
 data used for statistical analyses and figure generation. Only raw data
 corresponding to the representative images, traces, and gels shown in the
 figures are deposited; raw data from additional experimental replicates
 generated during the study are not included. ### Files and variables ####
 File: Raw_Data.zip **Description:** The Raw_Data.zip archive contains raw
 data files corresponding specifically to the representative images and
 traces displayed in the main and supplementary figures of the manuscript.
 This archive does not include all raw data generated during the study or
 all experimental replicates.\ \ The contents include raw confocal
 microscopy image files corresponding to the images shown in the figures,
 representative electrophysiological tip-recording raw traces displayed in
 the manuscript, uncropped scanning electron microscopy (SEM) images
 corresponding to SEM images shown in the figures, and uncropped RT-PCR gel
 images corresponding to gel images displayed in the manuscript. Only raw
 files associated with the representative data shown in the figures are
 provided. Quantitative analyses and biological replicates derived from
 these and additional raw data are provided in the source data files.
 Description of individual raw data files Raw data_Fig. 1A–O\ Raw confocal
 microscopy image files underlying Figure 1A–O, illustrating the anatomical
 organization, expression patterns, and overlap of gustatory receptor
 neurons in the labellum. Raw data_Fig. 2K, L, M, N\ Representative raw
 extracellular tip-recording traces underlying Figure 2K–N, showing
 neuronal responses of labellar taste sensilla to fatty acids and control
 tastants. Raw data_Fig. 3B\ Uncropped RT-PCR gel images underlying Figure
 3B, corresponding to the gel image displayed in the manuscript for gene
 expression validation. Raw data_Fig. 4A–D\ Raw confocal microscopy image
 files underlying Figure 4A–D, illustrating neuronal expression patterns
 and experimental manipulations affecting gustatory signaling. Raw
 data_Fig. 6A\ Representative SEM image of mosquito labellum each for
 dorsal and ventral view respectively. Raw data_Fig. 6E–G and  6I–K\
 Representative raw extracellular tip-recording traces underlying Figure
 6E–G, illustrating changes in gustatory neuron activity across
 experimental conditions. Raw data folders for supplementary figures\ \ Raw
 data_Supplementary Fig. 1A–C\ Raw confocal microscopy image files
 underlying Supplementary Figure 1A–C, providing additional anatomical and
 expression data supporting Figure 1.\ \ Raw data_Supplementary Fig. 2D, E,
 G, H, J, K, M, N\ Representative raw extracellular tip-recording traces
 underlying the indicated panels of Supplementary Figure 2.\ \ Raw
 data_Supplementary Fig. 3A, B, D, E, G, H\ Raw confocal microscopy image
 files underlying Supplementary Figure 3, providing additional neuronal
 labeling and expression analyses.\ \ Raw data_Supplementary Fig. 4E–H\
 Uncropped RT-PCR gel images underlying Supplementary Figure 4E–H.\ \ Raw
 data_Supplementary Fig. 7E\ Representative raw extracellular tip-recording
 traces underlying Supplementary Figure 7E. #### File: Source_Data.zip
 **Description:** The Source_Data.zip archive contains all quantitative
 source data used to generate the figures in the manuscript, provided in
 Excel format. These data include values derived from all biological
 replicates used for statistical analysis, even when only representative
 raw images or traces are shown in the figures.\ \ The archive contains two
 subfolders. The Excel_Sheets folder contains numerical source data used
 for plotting and statistical analyses. The Images folder contains
 uncropped but processed images used in Fig. 1A–O and Supplementary Fig.
 1A–C. Each figure has a corresponding Excel file labeled by figure name.
 For example, the source data file for Figure 2A is named
 Source_Data_Fig_2A.xlsx, and the file for Supplementary Figure 3A is named
 Source_Data_Supplementary_Fig_3A.xlsx. All experimental details are
 described in the Methods and figure legend sections of the manuscript.
 Description of individual source data files\ \ Source data files labeled
 Source Data_Fig. 2A–I contain data obtained from proboscis extension
 response (PER) assays performed in flies exposed to different chemical
 stimuli. Each value represents the response of an individual fly, with “1”
 indicating a positive PER and “0” indicating a negative PER. Rows
 correspond to experimental variables, and columns correspond to groups of
 flies. Each column represents one biological replicate (N = 1), consisting
 of a group of flies.\ \ The Source Data_Fig. 2J file contains data
 obtained from tip-recording assays performed on labellar taste hairs of
 flies exposed to the indicated concentrations of tastants. Each recording
 lasted 10 seconds, but only the first 1 second was analyzed. Rows indicate
 experimental variables, and columns represent individual flies used for
 recordings. Representative raw traces displayed in the manuscript are
 provided in the Raw_Data.zip archive.\ \ The Source Data_Supplementary
 Fig. 4C file contains data obtained from quantitative real-time PCR
 (RT-qPCR) experiments. PCR was performed using a Bio-Rad qPCR system, and
 Ct values are reported for each gene. Ribosomal protein S7 (RPS7) was used
 as the internal control. Uncropped RT-PCR gel images corresponding to
 those displayed in the manuscript are provided in the Raw_Data.zip
 archive.\ \ Source data files Source Data_Fig. 5B–C contain data obtained
 from a single blood-feeding assay using adult female mosquitoes. The
 values represent the percentage of mosquitoes engorged on blood feeders
 with or without chemical treatments. Rows indicate experimental variables,
 and columns represent biological replicates.\ \ Source data files Source
 Data_Fig. 5F–I contain data obtained from two-way choice blood-feeding
 assays using adult female mosquitoes. These files include the number of
 mosquitoes choosing either a control blood feeder or a blood feeder coated
 with fatty acids or other chemicals. Preference Index (PI) values were
 calculated from these counts. Rows indicate experimental variables, and
 columns represent biological replicates.\ \ Source data files Source
 Data_Fig. 6H and 6L contain data obtained from tip-recording assays
 performed on labellar taste hairs of female mosquitoes. Each recording
 lasted 10 seconds, but only the first 500 milliseconds were analyzed. Rows
 indicate experimental variables, and columns represent individual female
 mosquitoes used for recordings. Representative raw traces displayed in the
 manuscript are included in the Raw_Data.zip archive. Source data files
 Source Data\_ Supplementary Fig. 8\ Source data underlying Supplementary
 Figure 8, containing quantified calcium imaging responses from HEK293
 cells heterologously expressing Drosophila melanogaster Pain or Aedes
 aegypti Pain1 channels. The data represent normalized changes in
 intracellular calcium levels measured following stimulation, and were used
 to generate the plots shown in the supplementary figure. Rows represents
 cell used for the analysis. \ \ The Source_Data_Table_1 file contains data
 generated from confocal microscopy analyses. Immunohistochemistry was
 performed on fly labella with counterstaining to visualize distinct
 gustatory receptor neurons (GRNs), and samples were imaged using a
 confocal microscope. Neurons were counted to assess overlap between
 different GRN populations. Representative confocal raw images displayed in
 the figures are included in the Raw_Data.zip archive. Rows indicate
 variables, and columns represent the number of samples analyzed.\ \ Other
 source data Excel files for main and supplementary files follow similar
 formats and can be interpreted using the descriptions above. \ Acronyms:\
 LA: Linoleic acid\ HA: Hexanoic acid\ BA: Butyric acid\ DA: Decanoic acid\
 1 s: 1 second\ 500 ms: 500 milliseconds\ PER: Proboscis extension
 response\ TR: Tip recording\ WT: Wild-type\ PI: Preference Index\ Ave:
 Average of PI\ SEM: Standard error of mean ## Code/software Confocal
 microscopy data were acquired using a Zeiss LSM 900 confocal microscope.
 CZI files can be opened using ImageJ/Fiji or Zeiss ZEN software.
 Tip-recording data were acquired using AutoSpike software (version 3.1;
 Syntech;
 [https://www.ockenfels-syntech.com/download-2-2/](https://www.ockenfels-syntech.com/download-2-2/)) and require this software for viewing and analysis.