Data from: Divergent sensory transcriptomic profiles in positive and negative learning in Bicyclus anynana butterflies
Data files
Oct 29, 2025 version files 133.89 MB
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avap_activity_females.R
4.52 KB
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avap_ant_binarise_wgcna.R
13.21 KB
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avap_ant_genereads.R
11.72 KB
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avap_bargraphs_only.R
1.84 KB
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avap_br_binarise_wgcna.R
14.72 KB
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avap_br_genereads.R
10.33 KB
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avap_deg_boxplots.R
38.33 KB
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avap_ey_binarise_wgcna.R
13.22 KB
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avap_ey_genereads.R
10.19 KB
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avap_heatmaps_collated.R
14.45 KB
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avap_venn_diagram.R
7.08 KB
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dds_ant_all.RData
45.38 MB
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dds_br_all.RData
44.57 MB
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dds_ey_all.RData
43.12 MB
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perm_ant_ap_cont.csv
34.05 KB
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perm_ant_av_ap.csv
71.80 KB
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perm_ant_av_cont.csv
99.54 KB
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perm_br_ap_cont.csv
69.08 KB
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perm_br_av_ap.csv
72.96 KB
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perm_br_av_cont.csv
74.65 KB
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perm_ey_ap_cont.csv
106.50 KB
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perm_ey_av_ap.csv
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perm_ey_av_cont.csv
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README.md
7.06 KB
Abstract
Mate preference learning, where individuals learn to prefer or avoid specific phenotypes during mate selection, is pervasive across animal taxa and influences reproductive isolation and trait evolution. Despite its significance, the genetic basis underlying the associated valence attribution (whether to prefer or avoid) remains largely unclear. Both in terms of what genes are associated with attributing valence, and whether valence attribution is associated with transcriptional changes in both sensory tissues and neural circuits in the brain, or is restricted to the brain. Here, we investigate the neurogenomic basis of positive and negative mate preference learning using Bicyclus anynana butterflies. We put females in positive or negative learning scenarios, as well as a naïve control, and compared their transcriptomic profiles across three tissues: antennae, eyes, and brain using RNA-sequencing. Our results reveal tissue-specific transcriptional responses, with the antennae showing higher transcriptomic changes during negative learning and the eyes showing higher transcriptomic changes during positive learning, relative to the naïve control, indicating that valence attribution during learning may not be restricted to transcriptional changes in the brain. We identified a subset of genes in each tissue whose expression patterns changed with valence, as well as genes previously linked to classical conditioning pathways, supporting the hypothesis that imprinting-like learning and classical conditioning share molecular mechanisms. Our findings suggest that valence attribution in mate preference learning involves tissue-specific transcriptional responses in sensory and brain tissues, emphasising the role of peripheral sensory systems in modulating learned mate preferences.
Dataset DOI: 10.5061/dryad.j9kd51crw
Description of the data and file structure
Naïve females were isolated and were not exposed to training stimuli (i.e. males), females that were exposed to a randomly selected 4-spotted male with undisrupted male sex pheromones were considered given a “positive training exposure”, and females that were exposed to a randomly selected 2-spotted male with disrupted male sex pheromones were considered given a “negative training exposure” (n = 10 per training exposure).
After one hour of exposure (training), the females were decapitated with RNA-free scissors and flash frozen. Three tissues: antennae, eyes and brain, were dissected and their RNA was extracted. Libraries were prepared and sent for RNA-seq. Sequencing data was analysed using DESeq2 and permutation tests.
Files and variables
For all "perm_.csv" files:
- perm_ant_ap_cont.csv
- perm_ant_av_ap.csv
- perm_ant_av_cont.csv
- perm_br_ap_cont.csv
- perm_br_av_ap.csv
- perm_br_av_cont.csv
- perm_ey_ap_cont.csv
- perm_ey_av_ap.csv
- perm_ey_av_cont.csv
"perm_ant..." files refer to antennae data, "perm_br..." files refer to brain data, and "perm_ey..." files refer to eye data.
"ap_cont" refers to DEGs between positively-trained and naive females, "av_cont" refers to DEGs between negative-trained and naive females, and "av_ap" refers to DEGs between positively-trained and negatively-trained females.
Empty cells and NA cells are used interchangeably. Usually, NAs are populated from genome alignments (i.e. NA when B. anynana genome is BLAST-ed to D. melanogaster genome), while empty cells were populated from manual annotation from FlyBase (from author). Regardless, these csv files have been prefiltered for gene counts < 10 (including NA gene counts), and are viable DEGs. The NAs/empty cells represent the lack of complete mapping or unknown functions.
Column descriptions in all "perm_"files:
Variables
- SeqName: Gene ID for Bicyclus anynana based on v1.2's reference genome (e.g. BANY...)
- perc_5: 5% tail of the permutated data p-value distribution
- perc_1: 1% tail of the permutated data p-value distribution
- baseMean: average normalised count for each gene across all samples
- log2FoldChange: effect size estimate of gene expression change between two treatments
- lfcSE: standard error estimate for log2fc
- pvalue: p-value derived from DESeq2 analysis
- padj: p-adjusted value derived from DESeq2 analysis
- Description_c_1.2: Gene name after blasting B. anynana reference genome to NCBI using BLASTX
- Description_p_1.2: Gene name after blasting B. anynana reference genome to NCBI using BLASTP
- Name_dmel: Gene/ortholog name in D. melanogaster
- Symbol_dmel: Gene/ortholog ID in D. melanogaster
- Description_dmel: Similar to Name_dmel, except it has a RefSeq accession number prefix.
- Protein.accession_dmel: Protein accession number of D. melanogaster ortholog
- Locus.tag_dmel: Annotation symbol of D. melanogaster ortholog
- Description_dmel2: Very similar to Description_dmel, except this was based on common orthologs/genes after blasting B. anynana genome to D. melanogaster genome for exact ortholog blast hits.
- Gene.Type: "protein-coding" or not.
- Transcripts_accession_bany: Transcription accession number of B. anynana transcript
- Protein_accession_bany: Protein accession number of B. anynana transcript
- GO.IDs: GO IDs derived from BLAST2GO's mapping
- GO.Names: GO terms derived from BLAST2GO's mapping
- Accession_bany: RefSeq's accession number for B. anynana
- Drosophila_ortholog: Ortholog name in D. melanogaster for easy searching in FlyBase
- GO_terms_flybase: GO terms associated with ortholog based on FlyBase - more specific than BLAST2GO's mapping since this was done manually.
- comments: some comments regarding the functions of ortholog
- category: which gene category it was classified under (out of eight: learning and memory, vision, olfaction, mechanosensory, nervous system, neurotransmitter receptor, uncharacterised and other)
- dave_gene_list: Genes shared with Dave et al. (2023), as well as what was classified as "vision" or "chemosensory".
For all dds_.RData:
- dds_ant_all.RData
- dds_br_all.RData
- dds_ey_all.RData
dds_ant_all refers to RData containing DESeq data for antennae (i.e. dds file needed to replicate DESeq2 analysis)
dds_ey_all refers to RData containing DESeq data for eyes (i.e. dds file needed to replicate DESeq2 analysis)
dds_br_all refers to RData containing DESeq data for brain (i.e. dds file needed to replicate DESeq2 analysis)
Note: dds files were produced using the DESeq2 analysis/ codes below, with no modifications. Essentially, after producing these dds files, you can run all subsequent analyses (bargraphs, heatmaps, WGCNA, boxplots and venn diagrams). One can replicate all figures using these files.
Code/software
Software: R (or Rstudio), version 4.2.1+
Packages: DESeq2, dplyr, Rmisc, ggplot, WGCNA
All raw input files (.fastq) can be found in SRA Bioproject PRJNA1257361. The resulting ReadsPerGene.out.tab files are used as input files in the genereads.R script.
File: avap_ant_genereads.R
Description: Rscript for DESeq2 and permutation tests in antennae, for Pos, Neg and Pos-Neg comparisons
File: avap_ey_genereads.R
Description: Rscript for DESeq2 and permutation tests in eyes, for Pos, Neg and Pos-Neg comparisons
File: avap_br_genereads.R
Description: Rscript for DESeq2 and permutation tests in brain, for Pos, Neg and Pos-Neg comparisons
File: avap_activity_females.R
Description: Rscript for comparing activity levels of positively-trained females with naive females during exposure to male
Input file for activity level can be found in: Westerman, Erica; Ernst, David; Agcaoili, Gabrielle; Merrill, Abbigail (2023). Behavioral data for: A learning experience elicits sex-dependent neurogenomic responses in Bicyclus anynana butterflies [Dataset]. Dryad. https://doi.org/10.5061/dryad.612jm647d
File: avap_bargraphs_only.R
Description: Rscript for plotting bar graphs of DEGs across comparisons
File: avap_heatmaps_collated.R
Description: R script for plotting collated z-scored heatmaps
File: avap_ant_binarise_wgcna.R
Description: Rscript for WGCNA analysis in antennae
File: avap_br_binarise_wgcna.R
Description: Rscript for WGCNA analysis in brain
File: avap_ey_binarise_wgcna.R
Description: Rscript for WGCNA analysis in eyes
File: avap_deg_boxplots.R
Description: Rscript for plotting DEG boxplots
File: avap_venn_diagram.R
Description: Rscript for plotting venn diagrams of DEGs
Access information
Other publicly accessible locations of the data:
- Raw sequence data can be found in NCBI SRA's BioProject PRJNA1257361
To determine the gene expression associated with different types of social learning, we exposed female B. anynana to different training/ exposure treatments for one hour (n = 10 per treatment) from August to October 2022. Exposure assays were conducted one hour after sunrise. Every individual used in our exposure assays were size- and age-matched: females were Day 0, ensuring sexual immaturity (Costanzo and Monteiro 2007), and males were Day 3, when their sex pheromones are known to be attractive to females (Nieberding et al. 2012). Naïve females were isolated and were not exposed to training stimuli (i.e. males), females that were exposed to a randomly selected 4-spotted male with undisrupted male sex pheromones were considered given a “positive training exposure”, and females that were exposed to a randomly selected 2-spotted male with disrupted male sex pheromones were considered given a “negative training exposure”, based on prior studies (Westerman et al. 2012; Westerman and Monteiro 2013). After one hour of exposure (training), the females were decapitated with RNA-free scissors. Each head was placed into individual RNA-free 1.5ml LoBind tubes, immediately flash frozen in liquid nitrogen, and stored in -80⁰C until dissection. Sensory (antennae and eyes) and brain tissues were dissected from these females, RNA was extracted, libraries prepared, and sent for RNA-seq. Sequencing data was analysed using DESeq2 analyses and permutation tests.
