[This template readme file should be edited to be relevant to your dataset. The template proposes a basic set of information to be provided about a dataset. Sections 1-3 provide key information about the dataset and should be completed as fully as possible; Sections 4-5 provide information for interpretation and use of the dataset, and should be completed according to your judgement. Ask yourself in completing these sections: what information would the user of this dataset need in order to be able to understand it or replicate the results? Use of the README plain text format for dataset documentation is not required, and may not be suitable for longer or more detailed documentation. In these cases, or if preferred, you can use PDF or MS Word. Information provided here must correspond accurately with information provided in the dataset metadata record, e.g. the dataset title should match exactly, the same Creators should be listed, etc. The readme file should be saved with the name README_[Creator surname]_[Publication year]. The file name should not exceed 32 characters. Examples: README_Smith_2025.txt; README_Jones-etal_2025.txt. Text within square brackets is instructional and should be deleted from the final version of the readme.] 1. ABOUT THE DATASET -------------------- Title: Expression of Heat Shock Proteins and thermal sensitivity of male fertility across six Drosophila species Creator(s): Claire H. Smithson, Elizabeth J. Duncan, Steven M. Sait, Amanda Bretman Organisation(s): University of Leeds Rights-holder(s):Unless otherwise stated, Copyright 2025 University of Leeds Publication Year: 2025 Description: These are data on offspring counts and heat shock protein expression after mild heat shock in males of 6 species of Drosophila fruit flies. Cite as: Smithson, Duncan, Sait & Bretman (2025) Dataset for 'Expression of Heat Shock Proteins and thermal sensitivity of male fertility across six Drosophila species' University of Leeds. https://doi.org/10.5518/1736 Related publication: Smithson, Duncan, Sait & Bretman (2025) Dataset for 'Expression of Heat Shock Proteins and thermal sensitivity of male fertility across six Drosophila species' (In preparation) Contact: a.j.bretman@leeds.ac.uk 2. TERMS OF USE --------------- Copyright 2025 University of Leeds. Unless otherwise stated, this dataset is licensed under a Creative Commons Attribution 4.0 International Licence: https://creativecommons.org/licenses/by/4.0/.] 3. PROJECT AND FUNDING INFORMATION ---------------------------------- Title: [title of project] Dates: 1.10.19-30.06.24 Funding organisation: BBSRC Grant no.: BB/M011151/1, BB/W016753/1 4. CONTENTS ----------- File listing Data for Smithson et al 2025 Expression of Heat Shock Proteins and thermal sensitivity of male fertility across six Drosophila species.xlsx Offspring counts - number of offspring produced after mating with a control female Species species name Ltorder order of species from lowest (1) to highest (6) lethal temperature (LT) from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 TFLorder order of species from lowest (1) to highest (6) Thermal Fertility Limit (TFL) temperature from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 Difference_LT_TFL difference between LT and TFL from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 Temperature 24h exposure to control 23oC or 29oC Offspring_count number of offspring Sterile whether the male produced no offspring (0) or at least one (1) Hsp expression - relative gene expression of selected hsp genes at a benign control and elevated temperature Species species name Ltorder order of species from lowest (1) to highest (6) lethal temperature (LT) from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 TFLorder order of species from lowest (1) to highest (6) Thermal Fertility Limit (TFL) temperature from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 Difference_LT_TFL difference between LT and TFL from Parratt et al 2021 DOI:10.1038/s41558-021-01047-0 Tissue carcass or reproductive tract (testes, accessory glands and ejaculatory bulb) Temperature 30min exposure to control 23oC or 29oC Gene hsp gene name RE relative expression 5. METHODS ---------- 2. Methods Fly Maintenance and Species Identity We utilised laboratory stocks of six closely related cosmopolitan drosophilid species as used by Parratt et al. (2021). These species were chosen as they have diverse responses to thermal shock, despite shared broad latitudinal ecological range, in addition to having readily available published genomes, facilitating target gene identification and primer design. Flies were maintained as per Parratt et al. (2021) at 23°C in ambient humidity and a 12:12h light:dark cycle. Species were raised on one of four food types to minimise effects of nutritional stress (Parratt et al., 2021) (see supplementary methods) and transferred to new vials weekly. Adults for experimentation were collected from vials in which five males and five females were permitted to mate and lay eggs for 48 hours, then the larvae were left to develop fully at 23°C. Adults were sexed as virgins within eight hours of eclosion on ice anaesthesia and kept in single species and single sex groups of 10 until experimentation. Heat stress exposure and fertility assay At seven days post eclosion (14 days for D. hydei to account for delayed male sexual maturation), males were either kept at 23°C or exposed a mild thermal shock, in an incubator, at 29°C. Our aim was to capture rapid responses simulating a situation where the temperature climbs towards the high point of the day during a heatwave. Hence we chose 29°C as this surpasses the ambient temperature experienced within the laboratory, but is well within the reproductive and survival limits by at least 5°C for all six species (Parratt et al., 2021, van Heerwaarden and Sgro, 2021). For the fertility assay, flies were heat shocked for four hours (starting 1 h after lights on) following Parratt et al. (2021). Flies were allowed to recover overnight before being paired with an age-matched virgin female and allowed 24 hours to mate and lay eggs. The adults were then discarded, and the offspring were allowed to complete development at 23°C, after which they were frozen and counted. This gave both a measure of TFL (proportion of sterile males) and of the Thermal Sensitivity of Fertility (number of offspring) (Bretman et al., 2024) as a result of a heat shock in the adult stage. Gene expression analysis Males were exposed to 23 or 29°C for 30 minutes to capture initial gene expression changes (Lerman et al., 2003). Whole flies were then flash frozen in liquid nitrogen and stored at -70°C. Samples were divided into the reproductive tract (comprising the testes, accessory glands and ejaculatory bulb) and rest of the carcass on ice. Samples were then stored in groups of five pairs of reproductive tracts/ carcasses at -70°C. For each species and both temperature treatments, six biological replicates were used. Detailed methods for RNA extraction and cDNA synthesis, choice of reference genes, genes of interest, primer design and RT-qPCR are in the ESM. The expression of seven heat shock proteins was analysed: hsp23, hsp26, hsp27, hsp60A, hsp60B, hsp70Ba and hsp83, as all of these are expressed in the reproductive tract of male D. melanogaster (Michaud et al., 1997, Jagla et al., 2018). We then confirmed all other species had homologues of these genes (Supp mat S.1.21). We tested a panel of potential reference genes that are highly stable in the male reproductive system in D. melanogaster; actin (Act5c), α-Tubulin, elongation factor 1 (EF1), Ribosomal protein L32 (RpL32), TATA-Box Binding Protein (Tbp), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Ribophorin II (Rpn2) (Jagla et al., 2018, Michaud et al., 1997, Ponton et al., 2011, Kofler et al., 2011, Palmieri et al., 2014, Matta et al., 2011, Kim et al., 2019, Liao et al., 2018, Zhai et al., 2014). The stability of reference genes was compared using both the carcass and male reproductive tract under 23°C and 29°C across all six species. Reference gene analysis was performed using Normfinder (Andersen et al., 2004) in addition to bestKeeper and geNorm2 utilising the ‘ctrlGene’ package in R (Zhong, 2019) to rank the reference genes across species. The highest ranked reference genes for each species were utilised to normalise RT-qPCR analysis, hence we do not compare across species in a quantitative manner. Quantification of relative gene expression relative to the two best reference genes was performed using the Pfaffl method (Pfaffl, 2001, Hellemans et al., 2007). The Ct values for the samples were determined relative to the minimum value among the biological replicates, considering the efficiency of the primer in use. Statistical Analysis All statistical analysis was performed using R version 4.2.3 (R Core Team, 2023), with each species analysed separately. The proportion of males that were infertile was analysed by χ2 tests (with a continuity correction) and number of offspring (retaining those that produced 0 offspring) using Mann-Whitney U tests. We used GLMs to compare the effect of temperature on somatic/ reproduction tissue for each gene, hence there are 42 models (6 species x 7 genes). We have detailed statistical analysis and figures for each comparison (by gene, species and tissue) in the ESM. Relative gene expression was determined by dividing the target gene's expression by the geometric mean of the reference genes for each sample. Gene expression fold changes were compared per gene using a linear model with Quasipoisson distribution, with tissue and temperature as fixed factors. Prior to this, a standard Poisson model was assessed for dispersion. To simplify the full model, Analysis of Deviances (AOD) was employed, utilising F or χ2 tests as appropriate. The AOD procedure aimed to retain the model's descriptive power while eliminating non-significant terms, starting with the full model with interaction terms. Once the model selection process was completed, the selected model was compared to the null model using AOD. Post-hoc pairwise comparisons between groups were performed using Tukey tests, using the 'emmeans' package (Lenth, 2022). Differential gene expression in each species between body parts at the benign temperature was performed with Mann-Whitney U tests. BRETMAN, A., FRICKE, C., BAUR, J., BERGER, D., BREEDVELD, M. C., DIERICK, D., CANAL DOMENECH, B., DROBNIAK, S. M., ELLERS, J., ENGLISH, S., GASPARINI, C., IOSSA, G., LAGISZ, M., NAKAGAWA, S., NOBLE, D. W. A., POTTIER, P., RAMM, S. A., ROWE, M., SCHULTNER, E., SCHOU, M., SIMÕES, P., STOCKLEY, P., VASUDEVA, R., WEAVING, H., PRICE, T. A. R. & SNOOK, R. R. 2024. Systematic approaches to assessing high-temperature limits to fertility in animals. Journal of Evolutionary Biology, 37, 471-485. LENTH, R. V. 2022. emmeans: Estimated Marginal Means, aka Least-Squares Means. [Online]. Available: https://CRAN.R-project.org/package=emmeans. [Accessed]. PARRATT, S. R., WALSH, B. S., METELMANN, S., WHITE, N., MANSER, A., BRETMAN, A. J., HOFFMANN, A. A., SNOOK, R. R. & PRICE, T. A. R. 2021. Temperatures that sterilize males better match global species distributions than lethal temperatures. Nature Climate Change, 11, 481-+.