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Roundup causes embryonic development failure, alters metabolic pathways and gut microbiota functionality in non-target species

Citation

Orsini, Luisa (2020), Roundup causes embryonic development failure, alters metabolic pathways and gut microbiota functionality in non-target species, Dryad, Dataset, https://doi.org/10.5061/dryad.mcvdncjws

Abstract

Background: Research around the weedkiller Roundup is among the most contentious of the 21st century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests.We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of Glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes.

Results: We demonstrate that chronic exposure to ecologically relevant concentrations of Glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the ‘Reactome’, we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways and collagen degradation, responsible for the repair of wounds and tissue remodeling.

Conclusions: Our results show that chronic exposure to concentrations of Roundup and Glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure, and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services.

Methods

The impact of Glyphosate and of its commercial formula Roundup (Bayer) were quantified on D. magna by measuring changes in fitness-linked life history traits, genome-wide transcription and the microbiota. Four genotypes were used: LRV3.5_15, LRV3.5_1 and LRV13_2 were previously isolated from a shallow lake in Denmark 37,92,93; P-IT is a laboratory reference strain provided by the Institute of Ecosystem study, CNR Verbania, Italy. Host response was measured in multiple fitness-linked life history traits.

Prior to starting the exposure to Glyphosate and Roundup, the genotypes were acclimated and synchronized for two generations in common garden conditions (16:8 light: dark regime, 20±1 °C and fed 0.8 mg Carbon/L of C. vulgaris daily) to reduce interference from maternal and grandmaternal effects. After two generations in these conditions, clonal replicates aged 24-48 hour from the second or following broods were randomly assigned to the experimental exposures in which host phenotype, transcriptome and microbiome were assayed. Fitness-linked life history traits were measured in the time spanning an individual life cycle (until release of the second brood); genotoxic effect was measured with the Comet assay.

Size at maturity (distance between the head and the base of the tail spine), age at maturity (first time eggs were observed in the brood chamber), fecundity (total number of offspring released summing first and second brood), failed development of juveniles from first and second brood, and mortality were measured in the time spanning an individual life cycle (until release of the second brood) in control conditions (borehole water) and after chronic exposure to Glyphosate (1 mg/L) and Roundup (Bayer) (1 mg/L). For size at maturity, all animals were measured after releasing their first brood into the brood pouch using image J software (https://imagej.nih.gov/ij/index.html). Failed development was quantified as the percentage of dead or aborted embryos per genotype in the time spanning an individual life cycle as follows: (AE+DO)/(AE+DO+LO), where AE is Aborted Eggs, DO is Dead Offspring and LO is live Offspring (LO) 94. Mortality rates per genotype were calculated with a survival model fit via the psm function in the rms R package V.3.3 95. A separate model was fitted to each treatment, in which the day of mortality and the mortality event were combined as the dependent variables (e.g. censoring). All mortality curves were plotted using the survplot function form rms package in R v.3.3.3 95.

The genotoxic effect of Glyphosate and Roundup was measured using the comet assay 40. The essay uses a microgel electrophoresis technique, in which a small number of cells are suspended in a thin agarose gel on a microscope slide, lysed, electrophoresed, and stained with a fluorescent DNA-binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus toward the anode, which resembles the shape of a comet. We applied the comet essay to the haemolymph extracted from ten non-exposed 24-48 hours old juveniles, used as reference and on juveniles of the same age exposed for 72h (prior to last instar) to either Glyphosate or Roundup, following standard protocols 40. DNA damage was quantified on a minimum of 50 cells as the percentage of Tail Intensity (TI%). TI is the ratio between the total intensity of the tail and the total intensity of the comet (head and tail together) and is directly proportional to DNA damage 40.

Usage Notes

1. Suppa_etal_life history traits: StrainID, treatment, biological replicate, mortality, age at maturity, size at maturity, fecundity in first and second brood, measured as the total number of offspring released, percentage of failed development offspring in first and second brood measured after controlling for maternal effect are shown.

2. Suppa_etal_come assay: StrainID, treatment, biological replicate, number of cells counted (No cells), median tail intensity (%), mean tail intensity (%), and standard deviation (SD) are shown. The tail intensity refers to the DNA damage quantified on a minimum of 50 cells as the percentage of Tail Intensity (TI%). TI is the ratio between the total intensity of the tail and the total intensity of the comet (head and tail together) and is directly proportional to DNA damage.

Funding

Natural Environment Research Council, Award: NE/N016777/1