An integrated host-microbiome response to atrazine exposure mediates toxicity in Drosophila

Abstract

The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.

Fig. 1. Baseline microbiome composition in Drosophila melanogaster.

a Study design. b Rarefaction curves of the observed OTUs to assess species richness in Drosophila (orange; obtained from AdMMF at 4−8 days of age) and Mus musculus (purple; obtained from C57BL/6J fecal samples at 12 weeks of age). c Metagenomic sequence analysis to determine the bacterial species composition of the Drosophila gut microbiome isolated from 20 adult females at 21 days of age. The bar indicates relative abundance level colored at the species level as indicated in the key. d Age-dependent change in the distribution of the Drosophila gut microbiome based on 16S sequencing of fecal samples. For each of the 16S experiments, embryos were distributed between 25 bottles on chemically defined fly food. Four days post-eclosion flies were collected and the weight-equivalent of 250 flies were transferred to small cages (100 mm diameter × 150 mm) for the aging study. The food was replaced once daily. Bars indicate relative bacterial abundance colored at the family level as indicated in the key.

Fig. 2. Effect of atrazine and paraquat on survival and gut microbiome in Drosophila melanogaster.

Effects of exposures on host and microbiome. a Host survival curves in response to 5−20 mM atrazine exposures. LC50 at 48 h is 16 mM for females and 7 mM for males. For each dose, we used 30 males and 30 females in triplicate aged 4−5 days post eclosion. Error bars indicate the standard deviation across replicates. b Host survival curves in response to 6−40 mM paraquat exposures. LC50 at 48 h is 19 mM for females and 43 mM for males. For each dose, we used 30 males and 30 females in triplicate aged 4−5 days post eclosion. Error bars indicate the standard deviation across replicates. c Principal component analysis of microbial abundance levels in control, atrazine, and paraquat treated flies. The control 0 and 72 h groups are closer to each other than the atrazine and paraquat groups at 72 h. d Bar charts showing the relative abundance of bacterial families in control (n = 5), 2 mM atrazine treated (n = 3) or 8 mM paraquat treated (n = 3) flies at 72 h after treatment. response to atrazine and paraquat. Both herbicides show a reduction in the amount of the Rhodospirillales grp and an increase in the Lactobacillales group. e Log₂fold change at the genus level for bacteria significantly changed after treatment with atrazine (FDR < 0.06). Bacteria are color-coded at the order level. f Germ-free host survival curves in response to 5 and 7 mM atrazine exposure. For each dose, we used 30 males and 30 females in triplicate aged 4−5 days post eclosion. Error bars indicate the standard deviation across replicates.

Fig. 3. Effect of atrazine on host transcription in adult flies.

Transcriptional profiling of males and females raised conventionally or germ-free after exposure to atrazine. a Genes with differential expression after 72 h atrazine exposure with fold changes ≥ 3 (adjusted p ≤ 1E−06) (red). Conventionally reared (CR) germ-Free reared (GF). Genes are ordered on the chromosomes X, 2L, 2, 3L, 3R, and 4. All genes are shown in gray. b Anatomical analysis of the genes identified by DeSeq2 (a) using their organ-specific maximal gene expression to assign each gene to a single organ system. c Gene Ontology analysis using ClueGO in Cytoscape (p < 0.05) of genes differentially expressed (FC ≥ 1.5 and adjusted p ≤ 1E−06) after 72 h in 2.0 mM atrazine treated CR and GF flies compared to CR and GF untreated control flies, respectively.

Fig. 4. Acetobacter tropicalis partially rescues atrazine toxicity in germ-free flies.

a Survival curves of AdMMF GF flies exposed to 2 mM atrazine, 2 mM atrazine supplemented with A.tropicalis. Survival curves of untreated GF, 0.25% DMSO treated GF, and 2 mM atrazine treated CR flies are included as controls. All atrazine treated flies die by 15 days whereas those supplemented with Acetobacter tropicalis survive for at least 23 days following the curve of flies reared conventionally. For each condition, we used 15 males and 15 females in triplicate aged 4−5 days post eclosion. Error bars indicate the standard deviation of the proportion of surviving flies. b Atrazine degradation pathway. Acetobacter tropicalis genes in green are those with high similarity to genes found on a Pseudomonas sp. strain ADP, pADP-1 plasmid (LKAX01000023), AtzE (ATJ92156.1), BiuH (ATJ90877.1) AtzE (ATJ91605.1), and AtzF (ATJ90896.1) Dur1 (ATJ90895.1); genes in yellow, AtzA and AtzC have weak similarity to a number of putative candidate genes including N-ethylammeline chlorohydrolase (ATJ89361.1) and D-glutamate deacylase (ATJ89456.1). atzD is the only gene not having any putative orthologs in the atrazine degradation pathway; c principal component analysis of metabolic features detected in CR and GF flies at zero or 72 h after atrazine treatment. d Heatmap of putatively annotated metabolites clustered and grouped by KEGG pathway classifications. Note that z-scores correspond to the inverse normal transform of ranks, not a measure of significance—z-scores are used to improve visualization only. e FlyScape visualization of the metabolomics data in the context of metabolic reactions (24, 48, or 72 h) and transcriptional changes in adult female flies (72 h timepoint; adjusted p ≤ 0.01). The network includes only putatively annotated metabolites and genes identified in our analyses.