Project description:Glyphosate is one of the most widely used herbicides globally. It acts by inhibiting an enzyme in an aromatic amino synthesis pathway specific to plants and microbes, leading to view that glyphosate poses no risk to other organisms. However, there is growing concern that glyphosate is associated with detrimental health effects in humans, and an ever-increasing body of evidence suggests that glyphosate affects other animals including pollinating insects such as bees. Although pesticides have long been considered a contributing factor in the decline of wild bee populations most research on bees has focussed on demonstrating and understanding the effects (particularly sublethal ones) of insecticides. To assess whether glyphosate poses a potential risk to bees we characterised the changes in survival, behaviour, digestive tract proteome and microbiome in the bumblebee Bombus terrestris after chronic exposure to field relevant doses of glyphosate alone and as part of the commercially available product RoundUp Optima+®. Regardless of source, changes in response to herbicide exposure in important cellular and physiological processes in the digestive tract of B. terrestris were observed, with the abundances of proteins associated with oxidative stress regulation, metabolism, cellular adhesion, the extracellular matrix, and various signalling pathways being altered. Interestingly, endocytosis, oxidative phosphorylation, the TCA cycle, and carbohydrate, lipid, and amino acid metabolism were differentially altered depending on whether the exposure source was glyphosate AI or RoundUp Optima+®. In addition, RoundUp Optima+®, but not the active ingredient glyphosate, impacted fungal diversity in the digestive tract microbiota. Our research provides new insights into the potential mode of action and consequences of glyphosate exposure at the molecular and cellular levels in bumblebees and highlights issues with current regulatory measures involving commercial formulations of pesticides where the impact of the co-formulants on non-target organisms are generally overlooked.

Project description:Despite all debates about its safe use, glyphosate still is the most widely applied active ingredient in herbicide products with renewed approval in the European Union until 2033. Non-target organisms are commonly exposed to glyphosate as a matter of its mode of application, with its broader environmental and biological impacts remaining under investigation. Glyphosate displays structural similarity to phosphoenolpyruvate (PEP), thereby competitively inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), crucial for the synthesis of aromatic amino acids in plants, fungi, bacteria, and archaea. The majority of microbes, including the gut bacterium Escherichia coli (E. coli), possess a glyphosate-sensitive class I EPSPS, making them vulnerable to glyphosate's effects. Yet, little is known about glyphosate’s interactions with other bacterial proteins or its broader modes of action at the proteome level. Here, we employed a quantitative proteomics and thermal proteome profiling (TPP) approach, to identify novel protein binding partners of glyphosate in the E. coli proteome. Glyphosate exposure significantly altered amino acid synthesizing pathways, including increased abundance in shikimate pathway proteins, suggesting a compensatory mechanism. Extracellular riboflavin concentrations were elevated upon glyphosate exposure, while intracellular levels remained stable. Thermal proteome profiling indicated an effect of glyphosate on the thermal stability of certain proteins beyond the target enzyme EPSPS, including AroH and ProA. An elevated structural similarity between the substrates of the interaction candidates and glyphosate, similar to the competitive binding between PEP and glyphosate at the EPSPS, could be a reason for their interaction with the herbicide. Overall, glyphosate induced metabolic disturbances in E. coli, extending beyond its primary target, thereby providing new insights into glyphosate's broader impact on microbial systems.

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