# Novel female reproductive organ differentiates postmating transcriptional response to insemination versus arrival of sperm in bedbugs ## Description of the data and file structure ### Data archive **File:** `data.zip` This archive contains the data files used in the analyses described in the associated manuscript. The archive contains multiple `.csv`, `.tsv`, and `.rds` files. Missing values are represented either as blank cells or `"NA"`. ------------------------------------------------------------------------ # File descriptions ## Shared reference files The following files are used in multiple analysis scripts. ------------------------------------------------------------------------ ## `DAVID2entrez_geneID.txt` Mapping file used to convert **UniProt protein IDs to Entrez gene IDs** using the DAVID Bioinformatics Resources. Source: https://davidbioinformatics.nih.gov/ **Columns** Column Description ------------- --------------------------- `From` UniProt protein accession `To` Entrez Gene ID `Gene.Name` Gene name annotation **Species:** *Cimex lectularius* ------------------------------------------------------------------------ ## `cimex_signal_peps_combined.rds` List of *Cimex lectularius* proteins predicted to contain **signal peptides**, generated by combining predictions from: - Phobius\ - SignalP 6.0 The file contains UniProt protein IDs predicted to encode secreted proteins. ------------------------------------------------------------------------ ## `blast_chm_locations.csv` Chromosomal location of each gene inferred from **BLASTn searches against the *Cimex lectularius* chromosome-level genome assembly (Miles et al. 2025)**. **Columns** Column Description ----------- ----------------------------------------- `gene_id` Entrez gene ID `chm` Chromosome on which the gene is located The *Cimex lectularius* karyotype is: - **Males:** 2n = 26 + X₁X₂Y\ - **Females:** 2n = 26 + X₁X₁X₂X₂ ------------------------------------------------------------------------ ## `codeml_model0_full_results.csv` Evolutionary rate estimates calculated using **PAML codeml (model 0)**. **Columns** Column Description ---------- ------------------------------------------ `GeneID` Entrez gene ID `t` Total branch length across the phylogeny `S` Number of synonymous sites `N` Number of nonsynonymous sites `dN` Nonsynonymous substitution rate `dS` Synonymous substitution rate `omega` dN/dS ratio ------------------------------------------------------------------------ ## `N0.tsv` Hierarchical Orthogroup assignments generated using **OrthoFinder**. Input consisted of protein FASTA files for each species included in the analysis. **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `HOG` Hierarchical Orthogroup identifier `OG` Orthogroup identifier (deprecated) `Gene Tree Parent Clade` Phylogenetic clade defining the orthogroup ----------------------------------------------------------------------- Species included in the analysis: - Acyrthosiphon\ - Aedes\ - Callosobruchus\ - Chemipterus\ - Cimex\ - Drosophila\ - Gryllus\ - Rhodnius\ - Triatoma ------------------------------------------------------------------------ ## `FlyBaseAllGenes_2_Uniprot_2025_05_07.tsv` Gene ID conversion table downloaded from **FlyBase** for *Drosophila melanogaster*. Source: https://flybase.org/ **Columns** Column Description --------------------- --------------------------- `From` FlyBase gene identifier `Entry` UniProt protein accession `Protein names` Protein name annotation `Gene Names` Gene symbol `Length` Protein length `Gene Ontology IDs` Associated GO terms ------------------------------------------------------------------------ ## `cimex_go_ids.rds` Gene Ontology (GO) annotations for *Cimex lectularius* proteins downloaded from **UniProt**. **Columns** Column Description ------------- -------------------------------------------- `Accession` UniProt protein ID `GOID` Semicolon-separated list of GO identifiers ------------------------------------------------------------------------ # Reproductive biology datasets ## `cimex_secretome.csv` List of candidate **seminal fluid proteins (SFPs)** identified through proteomic analysis of male reproductive tissues (Garlovsky et al., in preparation). **Columns** Column Description ----------- --------------------------------------------- `Protein` UniProt protein ID `only_in` Tissue(s) in which the protein was detected `bias` Tissue expression bias ------------------------------------------------------------------------ ## `cimex_Matings_BM_2024.csv` Copulation duration data for individual females. **Columns** Column Description ---------------- ------------------------------------------- `ID` Unique female identifier `Date` Date of mating observation `Time_in` Time the mating pair was introduced `Cop_start` Start time of copulation `Cop_end` End time of copulation `Cop_duration` Copulation duration `Treatment` Experimental treatment assigned to female `Notes` Additional observations ------------------------------------------------------------------------ ## `sperm_pres.csv` Presence/absence observations of sperm in female reproductive tissues. **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `Ind_ID` Individual female identifier `Timepoint` Dissection timepoint (`unmated`, `0h`, `1h`, `3h`, `6h`, `24h`) `tissue` Tissue examined (`Mesospermalege` or `Lower reproductive tract`) `value` 1 = sperm present, 0 = sperm absent ----------------------------------------------------------------------- ------------------------------------------------------------------------ ## `repeated_counts.csv` Blind replicate sperm counts performed on microscopy images. **Columns** Column Description ------------- ------------------------- `photo_id` Unique image identifier `count_one` First sperm count `count_two` Second sperm count ------------------------------------------------------------------------ ## `sperm_counts_noduplicates.csv` Sperm counts in the mesospermalege at timepoints after mating. **Columns** Column Description --------------- ------------------------------------------------------ `ID` Unique female identifier `time_point` Dissection timepoint (`0h`, `1h`, `3h`, `6h`, `24h`) `sperm_count` Number of sperm counted in image `time_hours` Timepoint in hours `sperm_ml` Estimated total sperm in the mesospermalege ------------------------------------------------------------------------ # RNA-seq expression datasets ## `sb_count_matrix.csv` Gene count matrix generated from RNA‑seq data using the **nf-core/rnaseq pipeline** and imported into R using **tximport**. Samples include unmated male and female tissues. **Columns** Column Description ------------------------ -------------------------------- `gene_id` Entrez gene ID `SEX_TISSUE-REPLICATE` Gene counts for each condition Tissue codes: Code Tissue ------ -------------------------- H Head L Lower reproductive tract M Mesospermalege O Ovaries T Testes ------------------------------------------------------------------------ ## `salmon.merged.gene_tpm.tsv` Gene expression values in **Transcripts Per Million (TPM)**. **Columns** Column Description ------------------------ ---------------------------- `gene_id` Entrez gene ID `gene_name` Gene name `SEX_TISSUE_REPLICATE` TPM values for each sample ------------------------------------------------------------------------ ## `txi_pmr.rds` Time‑course RNA‑seq dataset for mesospermalege and lower reproductive tract samples. Generated from **nf-core/rnaseq** and imported into R using **tximport**. The R object contains: - abundances --- estimated gene abundances\ - counts --- estimated gene counts\ - lengths --- effective transcript lengths Sample names follow the format: `sampleID-timepoint-organ-replicate` ------------------------------------------------------------------------ ## `metadata_pmr.rds` Metadata describing the experimental design for the RNA‑seq timecourse experiment. ------------------------------------------------------------------------ # Protein interaction predictions ## `sfp_msl_results.csv` ## `lrt_sfp_results.csv` Predicted protein--protein interactions generated using **AlphaFold3 complex structure predictions**. Interactions were predicted between: - female reproductive tract proteins with predicted signal peptides\ - candidate seminal fluid proteins (SFPs) Signal peptides were trimmed prior to structure prediction. Reference: Abramson et al. 2024 Nature. **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `sfp_prot` SFP protein identifier `xxx_prot` Female protein identifier (`msl` = mesospermalege, `lrt` = lower reproductive tract) `iptm` Interface predicted TM-score `ptm` Predicted TM-score `ranking_score` AlphaFold model ranking score `fraction_disordered` Fraction of predicted disordered residues `has_clash` Whether steric clashes were detected `chain_iptm` Chain-specific ipTM score `chain_ptm` Chain-specific pTM score `chain_pair_iptm` Pairwise interface score `chain_pair_pae_min` Minimum predicted aligned error between chains `pcomb` Concatenated protein pair identifier ----------------------------------------------------------------------- ------------------------------------------------------------------------ # Code and software All analysis code is available on GitHub: https://github.com/MartinGarlovsky/Bedbug_RNAseq ------------------------------------------------------------------------ # Data access Raw RNA‑seq reads are available from the **Sequence Read Archive (SRA)**. **BioProject accession:** PRJNA1427960