DOI: 10.5061/dryad.76hdr7t4h
title: Data from: Direct and trans-generational effects of tetracyclines on the microbiome, transcriptome, and male mating behavior of the sheep blowfly Lucilia cuprina

description: Tetracyclines are broad-spectrum antibiotics widely used in agriculture,
 medicine, and research. However, they are associated with harmful side
 effects. In arthropods, parental exposure to tetracyclines has been linked
 to reduced health and fitness in untreated offspring. These
 trans-generational effects of tetracyclines could jeopardize the success
 of pest control programs that use tetracyclines to control gene
 expression. In this study, we investigated the transgenerational effects
 of two tetracyclines, doxycycline (DOX) and anhydrotetracycline (ATC), in
 the blowfly Lucilia cuprina, a significant pest of sheep. To simulate the
 rearing conditions of a transgenic male-only release program, blowflies
 were reared on standard diet alone, or on standard diet plus DOX or ATC,
 for three generations, then reared for an additional fourth generation on
 standard diet alone. We used behavioral assays, 16S amplicon sequencing,
 and mRNA sequencing to determine how DOX and ATC influenced male sexual
 competitiveness, microbiome composition and gene expression in the third
 and fourth generations. We found that three generations of DOX treatment
 led to lower sexual competitiveness in both third- and fourth-generation
 males. In addition, DOX and ATC shifted the composition of the blowfly
 microbiome and altered the expression of numerous mitochondria- and
 immunity-related genes in both generations. Our study supports an emerging
 body of evidence that tetracyclines exert not only direct but also
 trans-generational effects, and sheds light on the transcriptional and
 microbial responses to antibiotic exposure and removal. Our findings
 emphasize the need for pest control programs that use tetracyclines to
 evaluate the long-term effects of these
description: # Data from: Direct and trans-generational effects of tetracyclines on the
 microbiome, transcriptome, and male mating behavior of the sheep blowfly
 Lucilia cuprina
 [https://doi.org/10.5061/dryad.76hdr7t4h](https://doi.org/10.5061/dryad.76hdr7t4h) ## Description of the data and file structure This dataset contains additional files associated with the manuscript "Direct and trans-generational effects of tetracyclines on the microbiome, transcriptome, and male mating behavior of the sheep blowfly *Lucilia cuprina*.” Version history **2/20/2025:** ●      Added the following new files: ○      S1 Fig.pdf ○      S2 Fig.pdf ○      S3 Fig.pdf ○      S1 Table.docx ○      S2 Table.docx ○      S3 Table.docx ○      S4 Table.docx ○      S1 Data.xlsx ○      S3 Data.xlsx ●      Combined previous files (“ANCOMBC2_comps_pairwise_allgroups.csv”, “sample-metadata.csv”, “PD_pvals_pairs.csv”, “Evenness_pvals_pairs.csv”, and “Shannon_pvals_pairs.csv”) into one file, “S2 Data.xlsx”. ●      Combined previous files (“qPCR input data for R.csv” and “qPCR raw data and stats.xlsx”) into one file, “S4 Data.xlsx”. Description of the data and file structure **S1 Fig.pdf.** This PDF file displays a figure visualizing Principal Coordinates of Analysis (PCoA) of Robust Aitchison distance of the *L. cuprina* microbiome samples. Percent values on axes 1 and 2 indicate the proportion of total variation in the distance matrix explained by each axis.  **S2 Fig.pdf.** This PDF file displays a figure visualizing Principal components analysis (PCA) of the *L. cuprina *transcriptome samples grouped by sex and developmental stage. Percent values on axes 1 and 2 indicate the proportion of total variation in the distance matrix explained by each axis.  **S3 Fig.pdf.** This PDF file displays a figure visualizing the experimental design of the study.  **S1 Table.docx.** This .docx file displays a table showing the results of PERMANOVA analysis of beta diversity for the *L. cuprina* microbiome samples.  **S2 Table.docx.** This .docx file displays a table showing how many bacterial taxa were missing or differentially abundant in *L. cuprina* microbiome samples when comparing experimental to control groups. **S3 Table.docx**. This .docx file displays a table showing the results of a study testing different dosages of anhydrotetracycline (ATC) used to maintain a transgenic *L. cuprina* line with a tetracycline-repressible female-killing gene (DH1).  **S4 Table.docx.** This .docx file displays a table showing the primers used in this study. Primer sequences, target sites, and (for qPCR primers) amplification efficiencies and R2 values are indicated. ### Files and variables #### File: S1\_Data.xlsx **Description:** This .xlsx file contains 2 tabs consisting of the raw data and statistical analyses from male sexual competitiveness assays. ##### Variables * **Raw Data**: This tab contains the raw data from male sexual competitiveness assays with wild-type, antibiotic-treated G3 males and their untreated G4 offspring vs. untreated EF3A males. This tab also contains a table showing the baseline sexual competitiveness of untreated wild-type males vs. untreated EF3A males. * **Statistics**: This tab contains the output of statistical tests run on the raw data, including the Shapiro-Wilk test for normality, Bartlett’s test for equal variance, a Chi-square test of independence, a one-way ANOVA for detecting differences between group means and pairwise t-tests between individual groups with Benjamini-Hochberg correction for multiple comparisons. #### File: S4\_Data.xlsx **Description:** This .xlsx file contains 10 tabs consisting of raw qPCR data, metadata, and associated statistical analyses. ##### Variables * **Raw data and calculations:** This tab contains the raw qPCR measurements (i.e., Cq values) for the GST-1, 16S, and COXI genes for each of the 54 samples used in this study. The calculations used to produce the delta-delta Cq values and associated fold changes reported in the manuscript are also shown in this tab. * **R input for stats:** This tab contains the matrix of sample data (including metadata) and associated delta-delta Cq values for importation into R. * **p-values 16S adult comps:** This tab displays the p-values (adjusted for multiple comparisons using the Benjamini-Hochberg correction) from pairwise comparisons of delta-delta Cq values for the 16S gene across adult sample groups using an unpaired t-test.  * **p-values 16S larva comps:** This tab displays the p-values (adjusted for multiple comparisons using the Benjamini-Hochberg correction) from pairwise comparisons of delta-delta Cq values for the 16S gene across larva sample groups using an unpaired t-test.  * \**p-values COXI adult comps: **This tab displays the p-values (adjusted for multiple comparisons using the Benjamini-Hochberg correction) from pairwise comparisons of delta-delta Cq values for the COXI gene across adult sample groups using an unpaired t-test.  * \**p-values COXI larva comps: **This tab displays the p-values (adjusted for multiple comparisons using the Benjamini-Hochberg correction) from pairwise comparisons of delta-delta Cq values for the COXI gene across larva sample groups using an unpaired t-test.  * **t-values 16S adult comps -t -va**: This tab displays the t-values (test statistic) from pairwise comparisons of delta-delta Cq values for the 16S gene across adult sample groups using an unpaired t-test.  * **t-values 16S larva comps -t -va**: This tab displays the t-values (test statistic) from pairwise comparisons of delta-delta Cq values for the 16S gene across larva sample groups using an unpaired t-test.  * **t-values COXI adult comps -t -va**: This tab displays the t-values (test statistic) from pairwise comparisons of delta-delta Cq values for the COXI gene across adult sample groups using an unpaired t-test.  * **t-values COXI larva comps -t -va**: This tab displays the t-values (test statistic) from pairwise comparisons of delta-delta Cq values for the COXI gene across larva sample groups using an unpaired t-test.  ●      ***How to interpret NA values:*** A value of "NA" in a cell means "not applicable." For example, the value of the column "Sex" for larval samples is NA because larvae were not sexed in this study. Similarly, matrices of statistical output (p-values and t-values) contain NA values to avoid redundancy (showing the same comparison twice) and can be ignored.  #### File: S2\_Data.xlsx **Description:** This .xlsx file contains 7 tabs consisting of 16S microbiome sample metadata, ASV counts, taxonomic assignments, and output of statistical analyses. ##### Variables * **16S sample metadata**: This tab contains the metadata for the 16S samples, formatted for importation into R. * **ASV table**: This tab lists the ASV counts for each 16S sample. * **Taxonomy table with DNA sequences** This tab lists the taxonomic identity assigned to each ASV, as well as each ASV’s reference DNA sequence (“refseq” column). * **Alpha diversity**: This tab displays the corrected p-values from 3 measures of alpha diversity (Faith PD, Shannon diversity, and Pielou’s evenness index) across all sample groups (pairwise Wilcoxon rank sum test with Benjamini-Hochberg correction for multiple comparisons). * **PERMANOVA beta diversity**: This tab displays the results of PERMANOVA analyses of beta diversity with mixed-sex experimental groups and single-sex experimental groups. * **ANCOM-BC2 results (differentially abundant taxa)**: This tab displays the results of ANCOM-BC2 analysis of differentially abundant taxa in larvae, adult males, and adult females. * **ANCOM-BC2 results (structural zeroes)**: This tab displays the results of ANCOM-BC2 analysis of structural zeroes (i.e., missing taxa) in larvae, adult males, and adult females. #### File: S3\_Data.xlsx **Description:** This .xlsx file contains 32 tabs, consisting of transcriptome sample metadata, read mapping statistics, raw and normalized read counts, lists of differentially expressed genes (DEGs) for each experimental group, and lists of enriched Gene Ontology (GO) terms associated with the DEGs in each experimental group. ##### Variables * **RNAseq statistics**: This tab lists the number of reads mapped to different types of transcripts overall and, for each sample, the number and percentage of reads uniquely mapped to the genome. * **Metadata:** This tab contains the transcriptome sample metadata, formatted for importation into R. * **Raw read counts:** This tab displays the raw read counts for each transcriptome sample. * **Normalized (DESeq2) read counts**: This tab displays the normalized (via DESeq2) read counts for each sample. * **DEGs G3 DOX L:** This tab displays the DEGs identified in G3 DOX larvae samples relative to the control group of G3 H2O larvae samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G3 ATC L:** This tab displays the DEGs identified in G3 ATC larvae samples relative to the control group of G3 H2O larvae samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 DOX L:** This tab displays the DEGs identified in G4 DOX larvae samples relative to the control group of G4 H2O larvae samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 ATC L:** This tab displays the DEGs identified in G4 ATC larvae samples relative to the control group of G4 H2O larvae samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G3 DOX AM:** This tab displays the DEGs identified in G3 DOX adult male samples relative to the control group of G3 H2O adult male samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G3 ATC AM:** This tab displays the DEGs identified in G3 ATC adult male samples relative to the control group of G3 H2O adult male samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 DOX AM:** This tab displays the DEGs identified in G4 DOX adult male samples relative to the control group of G4 H2O adult male samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 ATC AM:** This tab displays the DEGs identified in G4 ATC adult male samples relative to the control group of G4 H2O adult male samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G3 DOX AF:** This tab displays the DEGs identified in G3 DOX adult female samples relative to the control group of G3 H2O adult female samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G3 ATC AF:** This tab displays the DEGs identified in G3 ATC adult female samples relative to the control group of G3 H2O adult female samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 DOX AF:** This tab displays the DEGs identified in G4 DOX adult female samples relative to the control group of G4 H2O adult female samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **DEGs G4 ATC AF:** This tab displays the DEGs identified in G4 ATC adult female samples relative to the control group of G4 H2O adult female samples. Genes are annotated using names from the Ensembl genome database for L. cuprina ([https://biit.cs.ut.ee/gprofiler/convert](https://biit.cs.ut.ee/gprofiler/convert)). Genes are ordered by log2 fold change (lowest to highest). Only statistically significant genes are displayed (adjusted p-value &lt; 0.05). Genes with low read counts (&lt;5 in majority of samples) were excluded. * **Enriched GO terms G3 DOX L**: This tab displays the enriched GO terms associated with the DEGs in G3 DOX larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).         * **Enriched GO terms G3 ATC L**: This tab displays the enriched GO terms associated with the DEGs in G3 ATC larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).         * **Enriched GO terms G4 DOX L**: This tab displays the enriched GO terms associated with the DEGs in G4 DOX larvae samples relative to the control group of G4 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).         * **Enriched GO terms G4 ATC L**: This tab displays the enriched GO terms associated with the DEGs in G4 ATC larvae samples relative to the control group of G4 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).         * **Enriched GO terms G3 DOX AM**: This tab displays the enriched GO terms associated with the DEGs in G3 DOX adult male samples relative to the control group of G3 H2O adult male samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms G3 ATC AM**: This tab displays the enriched GO terms associated with the DEGs in G3 ATC adult male samples relative to the control group of G3 H2O adult male samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms G3 DOX AF**: This tab displays the enriched GO terms associated with the DEGs in G3 DOX adult female samples relative to the control group of G3 H2O adult female samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).     * **Enriched GO terms G3 ATC AF**: This tab displays the enriched GO terms associated with the DEGs in G3 ATC adult female samples relative to the control group of G3 H2O adult female samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).     * **Enriched GO terms G4 DOX AM**: This tab displays the enriched GO terms associated with the DEGs in G4 DOX adult male samples relative to the control group of G4 H2O adult male samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms G4 ATC AM**: This tab displays the enriched GO terms associated with the DEGs in G4 ATC adult male samples relative to the control group of G4 H2O adult male samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms G4 DOX AF**: This tab displays the enriched GO terms associated with the DEGs in G4 DOX adult female samples relative to the control group of G4 H2O adult female samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).     * **Enriched GO terms G4 ATC AF**: This tab displays the enriched GO terms associated with the DEGs in G4 ATC adult female samples relative to the control group of G4 H2O adult female samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons).     * **Enriched GO terms upreg DOX G3 L**: This tab displays the enriched GO terms associated with the set of upregulated DEGs in G3 DOX larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms downreg DOX G3 L**: This tab displays the enriched GO terms associated with the set of downregulated DEGs in G3 DOX larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms upreg ATC G3 L**: This tab displays the enriched GO terms associated with the set of upregulated DEGs in G3 ATC larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). * **Enriched GO terms downreg ATC G3 L**: This tab displays the enriched GO terms associated with the set of downregulated DEGs in G3 ATC larvae samples relative to the control group of G3 H2O larvae samples. Enriched GO terms are ordered by adjusted p-value (lowest to highest). Only significantly enriched terms are displayed (adjusted p-value &lt; 0.05, Benjamini-Hochberg correction for multiple comparisons). ## Code/software The data files are best opened in Excel. The supplementary tables with Word and the supplementary figures with Adobe Reader ## Access information Other publicly accessible locations of the data: * The raw 16S sequence reads and associated metadata from this study are available at NCBI Sequence Read Library BioProjectID: PRJNA1118283 ([https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1118283/](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1118283/)) - The RNAseq reads and associated metadata from this study are available at NCBI Sequence Read Library BioProjectID: PRJNA1227982 ([https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1227982](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1227982)) * The R code used in this study can be accessed on GitHub ([https://github.com/AlexisKriete/tetracyclines-microbiome-transcriptome](https://github.com/AlexisKriete/tetracyclines-microbiome-transcriptome)