This README file describes the data and methods used in the study "Identification of two odorant receptors tuned to alarm pheromone in the honey bee Apis mellifera". The study investigated the responses of two odorant receptors, AmelOR109 and AmelOR136, to various pheromonal blends, individual compounds, and a panel of diverse odorants using heterologous expression in Drosophila melanogaster, coupled with transcuticular calcium imaging and single sensillum recordings (SSR).

Data Files and Sheets

The primary data generated in this study consists of electrophysiological recordings (SSR) and calcium imaging data. Specific data files are not explicitly named in the manuscript; however, the data are summarized in the figures and corresponding statistical analyses.

This data set contains multiple sheets, and each sheet contains individual-level measurements for each stimulus.Below is a description of each file and sheet.

A. Pheromonal Blends (ΔF/F0) – Calcium Imaging : This sheet contains calcium imaging responses (ΔF/F0) of AmelOR136 and AmelOR109 to a panel of pheromonal blends: Alarm pheromone, Aggregation pheromone, Brood pheromone, Queen mandibular pheromone (QMP), Queen retinue pheromone (QRP) and Waggle dance pheromone (Ethyl hexanoate and air are control stimulation).Associated manuscript figures: Figure 1A and Figure 1D.

B. Individual Compounds (ΔF/F0) – Calcium Imaging :This sheet includes time courses and mean ΔF/F0 responses to individual pheromonal compounds tested at 1000 µg. Associated manuscript figures: Figure 1C & 1F: Mean responses to individual compounds.

C. Specificity to 42 Odorants (spikes/s) – SSR: This sheet contains single sensillum recording (SSR) data for AmelOR136 and AmelOR109 exposed to 42 compounds across 6 chemical families. Associated manuscript figure: Figure 2A–B.

D. Specificity to Acetate Derivatives (spikes/s) – SSR:This sheet includes SSR data for 18 acetate derivatives, each tested at 1000 µg. Associated manuscript figure: Figure 3.

E. Dose–Response Curves (spikes/s) – SSR: This sheet provides dose–response recordings for four compounds tested at five doses (1, 10, 100, 1000, 2000 µg). Associated manuscript figure: Figure 5.

Data Description and Analysis

Calcium Imaging Data

dF-F0: Represents the change in fluorescence intensity. Calculated as (F(t) - F(0)) / F(0), where F(t) is the fluorescence intensity at time t, and F(0) is the average fluorescence intensity of the 5 frames immediately before odor stimulation (frames 11-15).

Single Sensillum Recording (SSR) Data

• Peri-stimulus Time Histograms: Generated by dividing the 15-second recording into 100 ms bins. The average number of spikes per bin was calculated for all odorants.
• Odor Response Window: Odor responses were observed between 6.3s and 8.1s, with a peak at 7s.
• Firing Rate Calculation: The firing rate during odorant stimulation was calculated by averaging the number of spikes within a 1.8-second window (6.3s-8.1s).
• Spontaneous Firing Rate: Measured during a 1.8-second window before stimulation onset.
• Response Quantification: The response intensity was calculated by subtracting the spontaneous firing rate from the firing rate during odorant stimulation. Units are in spikes per second (spike-s).

Additional Metrics

Lifetime Sparseness (S): A measure of receptor specificity, ranging from 0 (unselective) to 1 (maximally selective). Calculated using the responses to all SSR tested compounds. Formula provided in the manuscript.

Experimental Details

• OR Selection: ORs were selected based on their expression level in antennae and phylogenetic position, using published data (Jain and Brockmann, 2020; Brand et al., 2017; Karpe et al., 2016; Robertson and Wanner 2006).
• Heterologous Expression: AmelOR136 and AmelOR109 were expressed in ab3A OSNs of Drosophila melanogaster lacking endogenous receptors DmelOR22a and DmelOR22b.
• Fly Containment: Flies were secured in pipette tips, allowing antennae exposure.
• Olfactory Stimulation: Constant airflow of 3 L/min, with 500 mL/min redirected through stimulation cartridges containing filter paper soaked with 10 μL of odorant solution (100 μg/μL in mineral oil, unless otherwise stated). Control stimuli included mineral oil and ethyl hexanoate.
• Odorant Stimuli: Six pheromonal blends (alarm, queen mandibular, queen retinue, aggregation, waggle dance, brood), unitary compounds contained in these blends and a panel of 42 individual odorants (followed by a panel of 18 esters) were used. Specific compounds and ratios are listed in the manuscript.
• Explanation of Missing Values: Several reasons explain the presence of missing values (NA) in the dataset:

(1) Biological preparation stability: Maintaining the flies alive and physiologically stable throughout the entire stimulation sequence is not always guaranteed. Occasional instabilities or loss of signal during the experiment resulted in gaps in the dataset, recorded as NA.

(2) Compounds added later in the experimental pipeline: Some odorant compounds were not included in the earliest experimental batches and were introduced in later sessions. Individuals recorded before the addition of these compounds therefore have NA values for those specific stimuli.

(3) Acquisition-related issues: A few individuals exhibit NA values due to technical difficulties within the acquisition setup (e.g., unstable baseline, brief loss of recording, or mechanical issues). However, these individuals still produced reliable and biologically meaningful responses for other compounds. We therefore retained them in the dataset rather than removing entire recordings due to a limited number of missing values. NA thus indicates missing data due to biological or technical constraints, not hidden data.

• Statistical Analysis: Friedman ANOVA followed by Wilcoxon post-hoc tests were used to compare responses. Benjamini-Hochberg correction was applied for multiple comparisons. Dose-response data were analyzed using Generalized Linear Mixed-Effects Models (GLMM).

The analysis likely involved the following software and packages (although specific scripts are not provided with the dataset):

• R (version 4.0.0 or higher): For statistical computing and data visualization.

R Packages:

• \tidyverse (ggplot2, dplyr, tidyr, readr): For data manipulation, visualization, and reading data files. 
•  \ggpubr: For creating publication-ready plots. 
•  \rstatix: For statistical functions and post-hoc analyses. 
•  \xlsx: For working with Excel files. 
•  \lme4: For fitting generalized linear mixed-effects models (GLMM).
•  'rstatix': To perform 'pairwise_wilcox_test', and 'friedman_test'

Data Interpretation

The study identified AmelOR136 and AmelOR109 as receptors responsive to alarm pheromone components. AmelOR136 exhibited high specificity, primarily responding to isopentyl acetate. AmelOR109 showed a broader response profile, activated by multiple pheromone components and other odorants. Lifetime sparseness calculations supported the higher specificity of AmelOR136. Dose-response experiments were conducted to assess the sensitivity of AmelOR136 to its key ligands.

This README provides a summary of the data and methods. For questions regarding the data, please contact benjamin.andreu@sorbonne-universite.fr or see more contacts in the article.