DOI: 10.5061/dryad.mcvdncjws
title: Roundup causes embryonic development failure, alters metabolic pathways and gut microbiota functionality in non-target species

description: 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 determine safe use of chemicals, defined by outdated toxicity tests.We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.
description: The impact of Glyphosate and of its commercial formula Roundup
 (Bayer) were quantified on <i>D. magna</i> 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
 <sup>37,92,93</sup>; 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 <i>C. vulgaris </i>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) <sup>94</sup>. Mortality rates per genotype were calculated with a survival model fit via the psm function in the rms R package V.3.3 <sup>95</sup>. 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 <sup>95</sup>. The genotoxic effect of Glyphosate and Roundup was measured using the comet assay <sup>40</sup>. 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 <sup>40</sup>. 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 <sup>40</sup>.
description: 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.