Effects of the glyphosate-based herbicide roundup on C. elegans and S. cerevisiae mortality, reproduction, and transcription fidelity

Abstract

Glyphosate-based weed killers such as Roundup have been implicated in detrimental effects on single- and multicellular eukaryotic model organism health and longevity. However, the mode(s) of action for these effects are currently unknown. In this study, we investigate the impact of exposure to Roundup on two model organisms: Saccharomyces cerevisiae and Caenorhabditis elegans and test the hypothesis that exposure to Roundup decreases transcription fidelity. Population growth assays and motility assays were performed in order to determine the phenotypic effects of Roundup exposure. We also used Rolling-Circle Amplification RNA sequencing to quantify the impact of exposure to Roundup on transcription fidelity in these two model organisms. Our results show that exposure to the glyphosate-based herbicide Roundup increases mortality, reduces reproduction, and increases transcription error rates in C. elegans and S. cerevisiae. We suggest that these effects may be due in part to the involvement of inflammation and oxidative stress, conditions which may also contribute to increases in transcription error rates.

Figure 1

Comparison of Traditional and Rolling Circle Sequencing Comparison of Traditional and Rolling Circle sequencing amplification methods. The wavy line represents an mRNA sequence. The red star represents a legitimate error, the yellow square represents a reverse transcription error, and the green circle represents a sequencing error. Rolling Circle amplification allows self-comparison of the original mRNA sequence, meaning that any errors of non-cellular origin can be cleared from the final sequence, giving much higher accuracy in determining the true number and type of RNA errors than is produced by traditional sequencing methods.

Figure 2

Roundup C. elegans and S. cerevisiae Population Effects Depicts effects of Roundup exposure at glyphosate concentrations of 5%, 1%, 0.1%, and a 0% control lacking Roundup on C. elegans and S. cerevisiae population levels and mortality rates. p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***. A.) Depicts the percent of live C. elegans individuals observed following 1 hour of exposure to Roundup. LD50 attained at approximately 1% glyphosate. B.) Depicts effects of Roundup exposure on C. elegans individuals over four days. Live worms, dead worms, and censored worms were included in population survival and growth rates. C.) Depicts relative effects of Roundup exposure on S. cerevisiae population growth and survival rates estimated via OD600 readings. OD600 measurements taken over 6 timepoints at 60-minute intervals.

Figure 3

Roundup and pH C. elegans and S. cerevisiae RNA Polymerase II Transcription Landscapes Transcription landscapes depicting mRNA transcription errors produced by RNA polymerase II following C. elegans and S. cerevisiae treatment with Roundup at the glyphosate percentages 5%, 1%, 0.1%, and a 0% control, or exposure to extreme pH environments. A). S. cerevisiae treatment with Roundup. Error bars: Standard deviation. B). S. cerevisiae treatment with pHs 2.5, 5.5, and 8.5. Error bars: Standard Error. C). C. elegans treatment with Roundup. Error bars: Standard deviation. D). C. elegans treatment with pHs 3, 7, and 10. Error bars: Standard deviation. * indicates base-substitutions for which Roundup exposure significantly impacts the error rate (p ≤ 0.05).

Figure 4

Roundup C. elegans and S. cerevisiae Differential Expression Plots Heat plot of gene expression levels between A). 4 experimental replicates of C. elegans control 0%, and 2 experimental replicates exposed to 5% glyphosate. B). Between 2 experimental replicates of S. cerevisiae control 0%, and 2 experimental replicates exposed to 5% glyphosate. Horizontal labels indicate sample type and replicate, with the first number representing glyphosate percentage, and the number following the dash indicating replicates. (RStudio Team, 2020). C). WormBase Enrichment Analysis plot depicting the top 15 biological process phenotype terms significantly enriched in C. elegans in the control 0% vs. 5% glyphosate experimental condition. (Angeles-Albores et al., 2016). D). YeastEnrichr Enrichment Analysis plot depicting the top 15 biological process phenotype terms significantly enriched in S. cerevisiae in the control 0% vs. 5% glyphosate experimental condition. Combined score: log p-value * z-score. (Chen et al., 2013; Kuleshov et al., 2016).

Figure 5

Roundup C. elegans and S. cerevisiae ReviGo Plot ReviGo plot showing Molecular Functions (A), Cellular Components (B), and Biological Processes (C), significantly represented under the experimental conditions of A). S. cerevisiae and B). C. elegans control 0% vs. 5% glyphosate exposure. Bubble color corresponds to the relative p-value associated with each provided GO term. Bubble size corresponds to the relative associated LogSize value for each GO term. (Supek et al., 2011; Supek & Škunca, 2017).