Summary

In the context of the growing use of biofungicides, this project aims to characterize the tolerance acquisition of aquatic biofilms (periphyton and leaf litter) toward these emerging chemicals in comparison to synthetic ones. To this end, we will implement an untargeted metabolomics approach based on high resolution mass spectrometry allowing the integrative characterization of the exposure and the global molecular/biochemical response of the whole biofilm. Also, its use in parallel of usual structural and functional descriptors will support understanding of the mechanism/responses involved in the tolerance and its ecosystem consequences (ecosystem services), and the identification of molecular markers linked to these responses. To do so, exposure in controlled conditions to model (bio)-fungicides) will be implemented in order to first unravel the mechanisms of tolerance and then evaluated the influence of environmental confounding factors. Beyond the response of the whole biofilms, the response of their individual component (as mono-specific culture and co-culture) will be investigated to order to gain understanding of the contribution of microbial interaction and species acclimatation in the acquisition of tolerance.

Objectives and research hypothesis

MEMENTO aims to address the following overarching question: What is the tolerance of aquatic ecosystems to biofungicides compared to the current synthetic ones? To this end, we will address specific questions (Q), providing evidence about several hypotheses (H)

Q1. Which metabolic pathways and metabolites are involved in the tolerance of freshwater biofilms to chemical stress? H1. The metabolomic profile providing an integrative picture of the exposure and the molecular response, its combined use with tolerance measurements and chemometric tools should allow identifying candidate molecular markers and their signaling pathways associated with the tolerance to chemical stress. In particular, the measurement of tolerance based on the metabolomic response should provide a more comprehensive assessment of the status of the whole biofilm associated with the tolerance acquisition than usual endpoints targeting specific autotrophic or heterotrophic communities. Moreover, such phenotyping would provide clues about potential cost of tolerance (i.e. impact of energy metabolism associated to tolerance). Finally, the comparison of the metabolomics fingerprints associated to tolerance for different chemicals would highlight potential co-tolerance mechanism (i.e. shared mechanism).

Q2. What is the link between molecular, structural, and functional responses at the community level during the acquisition of tolerance and what is the sequence of events along this acquisition? H2. While antifungal agents should trigger mainly alteration of fungal communities, the replacement of synthetic fungicides by wide spectrum pesticides such as copper should impair all the components of the biofilm. Hence, the metabolomic response should be different depending on the type of biofilm (i.e. autotroph-dominated in periphyton, heterotroph-dominated in decaying leaf litter). The parallel assessment of the molecular, structural, and functional responses in microbial communities should allow identifying the signaling pathways involved in the shift of the structure and/or the function associated with the tolerance to chemical stress. Also, such assessment along a kinetic of exposure should provide evidence whether structural responses appear before or after molecular and functional responses. Finally, the characterization of both the metabolome of the communities and of isolated species and co-culture would provide evidence about chemically-mediated microbial interaction involved in the tolerance acquisition.

Q3. Are there molecular markers (metabolites) specific to tolerance acquisition? H3. Since mechanisms of action can differ between (bio)fungicides, the strategy to cope with such agents might be different in the biofilm as well as the associated metabolomic response. Exposure in controlled conditions to various antifungal agents, used individually or in mixture should provide a way to identify relevant generic vs specific candidate biomarkers related to the tolerance. In addition, the characterization of confounding factors in controlled conditions should provide knowledge about their specificity to chemical stress and further use as potential biomarkers. Overall, this would also highlight co-tolerance phenomena (i.e. when exposure to one/several chemical(s) led to tolerance to another one). Altogether these investigations will provide a better understanding of tolerance acquisition by aquatic biofilms towards (bio)fungicides and their consequences on major ecosystem processes (i.e. primary production, respiration, nutrient cycling) (Objective 1, O1). Beyond this fundamental objective, we will identify molecular descriptors as candidate biomarkers able to detect tolerance to (bio)fungicides at the community level and thus preservation of investigated ecosystem functions (O2).

Expected results

MEMENTO will contribute to filling the gaps of scientific knowledge about:

• The molecular (i.e. metabolic) pathways and associated metabolites involved in the response, tolerance acquisition and tolerance itself of aquatic microbial communities and so ecosystems to bio vs synthetic fungicides
• The link between exposure to bio vs synthetic fungicides and molecular, physiological/functional and structural responses of aquatic micro-organisms/communities
• The influence of environmental factors on the responses and acquisition of tolerance of aquatic microbial communities to bio vs synthetic fungicides
• The co-tolerance and cost of tolerance theories related to the exposure to (bio)fungicides and environmental mixture in the global change context By doing so, MEMENTO will support:
• The definition/characterization of both Tolerance Outcome Pathways vs Adverse Outcome Pathways (i.e. the sequence of events, signaling pathways) at the community level related to bio vs synthetic fungicides exposure contributing to the mechanistic understanding of the community tolerance induced by chemical stress in the global change context.
• The identification of specific and sensitive molecular descriptors (i.e. metabolites or metabolite fingerprints) as candidate biomarkers able to early detect the tolerance/impact at the community level and preservation/alteration of their function and associated services.
• The use of biofungicides in the best practice way through the characterization of their ecotoxicity at the microbial community level.

Impact of the project

MEMENTO aims to characterize the tolerance of aquatic ecosystems to biofungicides, a class of emerging concern regarding their increasing use in the frame of agro-ecology. By doing so, it will have impacts on several scientific fields as well as economic, social and cultural field at short, medium and long term. At short term (within the project), MEMENTO will enhance knowledge in various scientific domains including ecotoxicology and ecology about the link between exposure to bio vs synthetic fungicides and molecular, physiological, structural and functional responses in aquatic microbial biofilms. This will support the characterization of both tolerance outcome pathways and adverse outcome pathways at the community level and the identification of molecular pathways and metabolites involved in the tolerance and preservation of their ecosystem functions and associated services. Thus, our findings will contribute to the discovery of candidate ecosystems biomarkers to monitor the tolerance of aquatic ecosystems in global change context. This could have socio-economic impact at longer term (see below). Even at short term, the project would have a societal/cultural impact through our participation to scientific days allowing to communicate towards the public, as well as regulatory impact in water policies through the organization of mid-term and final project meeting with stake-holders and policy makers. At medium term (5 years post project), MEMENTO will still impact the scientific community through new scientific publications and sharing of data on internal and external data repositories and online databases/libraries. In addition, our findings will draw scientific perspectives and open the opportunity for new scientific projects about the identification of the active components inside the biofungicides mixtures, the community tolerance to other chemical and environmental stress, and the further validation of existing metabolomics ecosystem biomarkers at large scale and the discovery of new ones for monitoring the sustainability of ecosystem services. Then, by providing new knowledge about the benefit/risk of biofungicides the project would have a socio-economic impact by taking into account other environmental constraints in the frame of agro-ecology development. At long term (10 years post project), MEMENTO results would be still available for further exploitation through data repository and online libraries. Also the further validation of the identified candidate biomarkers at larger scale (other agricultural, other climate) would support their implementation in the water bodies’ regulation (Water Framework Directive).