Additional file 1 of Chronic toxicity testing including transcriptomics-based molecular profiling in Cloeon dipterum
Creators: name: Germing, Kirsten
name: Ayobahan, Steve U.
name: Reinwald, Hannes
name: Vogt, Maria
name: Ringbeck, Benedikt
name: Göckener, Bernd
name: Eilebrecht, Elke
name: Kosak, Lena
name: Eilebrecht, Sebastian
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Composition of nutrient solution for Navicula pelliculosa culture. Table S2. Water parameters of the long- and short-term exposure tests. Table S3. Time-weighted mean concentrations of fipronil of the long-term exposure test. Table S4. Time-weighted mean concentrations of fipronil of the short-term exposure test. Table S5. Percentage of larvae ≥ L4 per replicate and treatment after three days exposure in the long-term exposure toxicity test with C. dipterum. Table S6. Percentage of larvae ≥ L6 per replicate and treatment after seven days exposure in the long-term exposure toxicity test with C. dipterum. Table S7. Percentage of larvae ≥ L7 per replicate and treatment after ten days exposure in the long-term exposure toxicity test with C. dipterum. Table S8. Percentage of larvae ≥ L4 per replicate and treatment after three days exposure in the short-term exposure toxicity test with C. dipterum. Table S9. Percentage of larvae ≥ L6 per replicate and treatment after seven days exposure in the short-term exposure toxicity test with C. dipterum. Figure S1. Left. C. dipterum larva in larval stage L3; Right. C. dipterum larva in larval stage N1. Figure S2. Larval development stages based on wing pad development of C. dipterum (adopted from Cianciara 1976). Figure S3. Molecular sturcture of the test substance fipronil. Figure S4. RNA-Seq read count normalization using DESeq2. (A) Raw read counts (left) and relative log expression (RLE) normalized read counts (right) of adult C. dipterum exposed to low (75.0 nμg/L) and high (300 ng/L) concentrations of fipronil and corresponding controls. Biological replicates are numbered as shown. Figure S5. Distributions of p-values, p-value conversion, and distributions of log(2)-fold changes upon low and high levels of fipronil exposure in adult C. dipterum observed by gene expression data in contrast to the control. Distribution of p-values for all genes after exposure to (A) low (75.0 n/L) and (B) high (300 ng/L) fipronil concentrations (Wald's t-test) (top). Wald's p-values were obtained and compared to converted p-values for multiple testing using Benjamini–Hochberg, as well as the corresponding p-values for exposure to low (75 ng/L) and high (300 ng/L) concentrations of fipronil (Middle). The distribution of log(2)-fold change values for all genes following fipronil exposure at low (75.0 ng/L) and high (300 ng/L) concentrations (bottom). For each case, the dotted line denotes the log(2)-fold change cut-off. Figure S6. MA-plot (top) showing log(2) transformed mean expression (gene count) to apeglm shrunk log(2)-fold change for every expressed gene in (A) Low and (B) High exposure condition in comparison to the control and Volcano plot (bottom) depicts the log(2)-fold change (lfc) values against the corresponding –log(10)(padj) values of genes that were differentially expressed after (A) Low and (B) High fipronil exposure to adult C. dipterum. The lfc value cut-off as well as the padj cut-off are indicated as dotted lines. Genes applying to both of them are colored red. Black dots represent genes with no significant difference. Figure S7. PCA (A) and t-distributed stochastic neighbor embedding (t-SNE) (B) of adult C. dipterum exposure to low (75.0 ng/L) and high (300 ng/L) concentrations of fipronil, as showed by RNA-Seq. On top of each panel, the total number of normalized genes used for clustering is displayed. Figure S8. Sample distance matrix analysis of adult C. dipterum exposed to fipronil at low (75.0 ng/L) and high (300 ng/L) concentrations, as well as the corresponding controls. The conditions are denoted by a color code. Figure S9. RNA-Seq read count normalization using DESeq2. (A) Raw reads (left) and Relative log Expression (RLE) normalized read counts (right) of C. dipterum larvae exposed to low (75.9 ng/L), mid (1500 ng/L), and high (300 ng/L) concentrations of fipronil, as well as the corresponding controls. Biological replicates are numbered. Figure S10. Distributions of p-values, p-value conversion, and distributions of log(2)-fold changes following low, mid, and high exposure of C. dipterum larvae to fipronil as observed by gene expression data in comparison to the control. Distribution of p-values for all genes following exposure to (A) low (75.0 ng/L), (B) mid (150 ng/L), and (C) high (300 ng/L) concentrations of fipronil (Wald's t-test) (top). Obtained Wald's p-values in comparison to converted p-values for multiple testing following Benjamini–Hochberg, as well as the corresponding p-values for exposure to low (75.0 ng/L), mid (150 ng/L), and high (300 ng/L) concentrations of fipronil (Middle). Distribution of log(2)-fold change values of all genes after exposure to fipronil at low (75.0 ng/L), mid (150 ng/L), and high (300 ng/L) concentrations (bottom). The dotted line represents the log(2)-fold change cut-off for each condition. Figure S11. MA-plot (top) showing log(2) transformed mean expression (gene count) to apeglm shrunk log(2)-fold change for every expressed gene in (A) Low, (B) Mid and (C) High exposure condition in comparison to the control and Volcano plot (bottom) depicts the log(2)-fold change (lfc) values against the corresponding –log(10)(padj) values of genes that were differentially expressed after (A) Low, (B) Mid and (C) High fipronil exposure to C. dipterum larvae. The lfc value cut-off as well as the padj cut-off are indicated as dotted lines. Genes applying to both of them are colored red. Black dots represent genes with no significant difference. Figure S12. PCA (A) and t-distributed stochastic neighbor embedding (t-SNE) (B) of samples upon exposure to low (75.0 ng/L), mid (150 ng/L), and high (300 ng/L) concentrations of fipronil to C. dipterum larvae as showed by RNA-Seq. On top of each panel, the total number of normalized genes used for clustering is displayed. Figure S13. Sample distance matrix analysis of C. dipterum larvae samples exposed to fipronil at low (75.0 ng/L), mid (150 ng/L), and high (300 ng/L) concentrations, as well as the corresponding controls. The conditions are denoted by a color code. Figure S14. Chromatogram of fipronil (quantifier and qualifier mass transition) of the control sample at day 21 (fresh) of the chronic test. The measured concentration in this sample was 0.018 μg/L (