Small freshwater reservoirs are ecosystems of global importance both for the great biodiversity they shelter, the ecosystem services they provide and the freshwater resources they constitute. An increase in temperatures and precipitation intensity due to climate change is expected by the end of the century. These changes should result in an increase in interactions between lakes and watersheds, leading in particular to increased inputs of terrestrial organic matter (tDOM) likely to impact the functioning of small lake ecosystems.

In this context, a mesocosm experiment was carried out to study the combined effect of two disturbances - input of tDOM and temperature rise - on pelagic communities. The experiments were conducted within the National Experimental Platform for Aquatic Ecology (PLANAQUA) at CEREEP-Ecotron Ile-de-France located in Saint-Pierre-lès-Nemours (France). The experiment was carried out in twelve 1m³ mesocosms placed outside during 6 weeks, from June 8 to July 19, 2021. The mesocosms are circular tanks with walls allowing the circulation of a heat transfer fluid whose temperature is controlled by thermo-refrigerator pumps. The mesocosms were randomly filled with unfiltered water taken from small oligotrophic tanks located on the site and containing plankton and microorganisms. Four juvenile fish roach (Rutilus rutilus) were introduced at the start of the experiment in each mesocosm. Three replicates of two crossed treatments were carried out in these mesocosms. These treatments included a temperature differential of 6°C (18°C and 24°C) maintained throughout the experiment and, for the addition of tDOM, either no addition or an initial increase in tDOM concentration of 65 µM dissolved organic carbon originating from natural forest soil, representing a total enrichment in organic carbon of +25%. The mesocosms were manually stirred every day before sampling and measurements with different probes (see below for details).

The mesocosms (called ECORIUM) are made of double-walled 316 stainless steel tanks. The internal diameter is 1300 mm and the height is 820 mm. Glycol water circulates within the double wall to regulate the temperature inside the tank between -10°C and 45°C.

The three in situ control mesocosms were labeled C1, C2, C3, the three mesocosms in which terrestrial runoff was simulated were labeled T1, T2, T3, the three mesocosms in which temperature warming was simulated were labeled W1, W2, W3, the three mesocosms in which both terrestrial runoff and temperature warming were simulated were labeled TW1, TW2, TW3.

• Sampling was done using a 2-L sampling bottle (Uwitec Sapling Bailer USB 50020, Mondsee, Austria).
• Immersion of a multiparameter probe (YSI EXO2) was performed after stirring to measure the temperature, conductivity, turbidity, salinity, pH, O₂ saturation and Chlorophyll a values in surface waters.
• Immersion of a multispectral spectrofluorometer probe (FluoroProbe BBE Moldaenke) was performed after stirring the water in the mesocosms to measure the concentrations (in µg Chl-a.L^-1^) of different phytoplanktonic groups: green algae, cyanobacteria, diatoms and cryptophytes. Yellow substances and transmission (in %, as a proxy of the turbidity) were also measured.

For the analysis of inorganic nutrients (PO₄^2-, NO₃^-, ^NO₂-^) water samples (30 mL) were filtered through 0.2 μm filters, stored in tubes at -20°C, and then analysed using an autoanalyser (AA3HR, Seal Analytical).

• For dissolved organic carbon (DOC) concentrations, samples (30 mL) were filtered through pre-calcined GF/F glass fiber filters with a diameter of 47 mm (Whatman), then stored in glass tubes and preserved with 35 μL of 85% H₃PO₄ at room temperature and in the dark. DOC concentrations were then determined using the high catalytic oxidation technique with a Shimadzu TOC-L analyser. For optical properties of dissolved organic matter (DOM), 60 mL of each sample was filtered through a 47 mm diameter GF/F filter and collected in an amber glass tube. All samples were stored at 4°C in the dark prior to characterization. Absorbance measurements of colored DOM (CDOM) were performed using a spectrophotometer HP8453 (Hewlett–Packard, Agilent Technologies, Massy, France). Fluorescence measurements of DOM (FDOM) were performed using a Varian Cary Eclipse spectrofluorometer according to Murphy et al. (2013). Data were analysed using three-dimensional excitation-emission matrix fluorescence spectroscopy (EEM) and parallel factor analysis (PARAFAC). The emission and excitation characteristics allowed compounds to be identified into two categories: humic substances with humic acids (Component 3) and fulvic acids (Component 5), and substances of protein origin with aromatic amino acids such as tyrosine (Component 1) and tryptophan (Component 4). The nature of Component 2 (protein-like compound) has not been identified. Indices were calculated to characterise the organic matter composition, including the BIX biological index indicating recent indigenous contributions (Huguet et al., 2009), the HIX humification index providing information on the source, degree of humification and molecular structure, the fluorescence index FI, which provides information on the source or degree of organic matter degradation (Fellman et al., 2010), and the a254 index, which indicates the content of aromatic amino acids (D’Andrilli et al., 2022).
• Samples (70 to 170 mL) for particulate organic carbon (POC) and nitrogen (PON) concentrations (mg.L^-1^) were filtered onto pre-calcined GF/F glass fiber filterswith a diameter of 47 mm (Whatman). In order to remove the mineral fraction from filters, 550 μL of H₂SO₄ was added to the filters, which were then dried at 50°C for several hours. The concentrations of carbon and nitrogen were then determined after combustion through a CHN elemental analyser (Flash EA 1112 Series, Thermo Electron Corporation) according to Sharp (1974).

• Samples for bacterial abundance (1.5 mL) were preserved with glutaraldehyde (1% final concentration) and stored at -80°C. Samples were stained with SYBR^TM Green I (Thermofisher Scientific) and analyzed on a flow cytometer (FACSCanto, BD Biosciences) according to Gasol and Del Giorgio (2000).
• Samples for phytoplankton abundance (1.5 mL) were preserved with glutaraldehyde (1% final concentration) and stored at -80°C. Samples were analyzed on a flow cytometer (FACSCanto, BD Biosciences). Populations were identified and enumerated based on forward scatter (FSC), side scatter (SSC) light diffractions, and natural pigment fluorescence (chlorophyll-a, phycoerythrin). This allowed the discrimination of two groups of picophytoplancton (PicoEuk1 and PicoEuk2), and one group of nanophytoplankton (Nano).

Respiration rates were derived from continuous dissolved oxygen (O₂) measurements using a SensorDish Reader (SDR; Presens, Germany) optical sensing system equipped with 24 glass vials of 5 mL containing non-invasive O₂ sensors (OxoDish). Incubations were performed at in situ temperature (18°C or 24°C) in the dark in climatic chambers.

Extracellular aminopeptidase activity was measured with the use of a fluorogenic substrate (Hoppe, 1983) with the modifications for plate readers described in the study by Sala et al. (2016). Incubations were performed at in situ temperature (18°C or 24°C) in the dark in climatic chambers.

The metabolic profile of microbial communities was estimated using the Biolog® EcoPlate™ method according to Garland et al. (2001). At some selected sampling dates, raw water (150 µL) was inoculated in each well of the microplates (Alliance Bio-expertise, Guipry-Messac, France). Microplates were then incubated in the dark either at 18°C or at 24°C, depending on the temperature of the mesocosm from which the water samples were taken. Absorbance was measured at 590 nm every 24h during 9 days using a Microplate Reader (Bio-Rad Laboratories, Model 680). After data trimming (absorbance-threshold value of 0.10), the average well-colour development (AWCD) was determined for the whole microplate, per substrate family and per substrate. The functional richness (FR) was expressed as the number of different substrates used by the microbial community. The NUSE index (Nitrogen Use Index) was expressed as the relative absorbance of Nitrogen-containing substrates (Sala et al., 2006). The Shannon diversity and Pielou evenness indices were calculated. All data used were acquired after 4 days of incubation, to ensure that all enzymatic reactions were detected.

Zooplankton were collected on the following dates: 08/06, 11/06, 15/06, 21/06, 28/06, 05/07, 12/07 and 19/07. Samples were obtained by filtering 50 μm of water through a nylon filter, collected using a Niskin bottle for each mesocosm. A total of 27.44 L of water was filtered per mesocosm (28 L collected minus samples taken for phytoplankton and protista analysis). The zooplankton collected were transferred to a polypropylene vial stored in 80% ethanol at 4°C in the dark.

Prior to analysis, the samples were refiltered into 3 categories: greater than 850 μm, between 850 μm and 400 μm and less than 400 μm. The first category was processed using a binocular magnifier, while the other two were processed using the Flowcam 8100 (Beckman Coulter) and Ecotaxa software (Picheral et al., 2017). The Flowcam is an imaging tool used to classify and measure organisms and particles contained in a liquid medium. Before being run through the Flowcam, each sample was diluted in distilled water to obtain 500mL and 5 mL were taken using a propipette and then placed on the device for analysis. The images generated by Flowcam were sorted and determined using the Ecotaxa online platform. The results are expressed in nb/L and have been grouped into functional groups: (1) Large filter feeders corresponding to Ostracods and Cladocerans; (2) Small filter feeders corresponding to Rotifers and Nauplii; (3) Predators corresponding to Copepods except Nauplii.

Four fish fry (Rutilus rutilus) were introduced into each mesocosm at the start of the experiment. The average biomass of the fry introduced at the start of the experiment was calculated by weighing 32 fish from the same batch as those used to inoculate the mesocosms, thus avoiding handling and injuring the individuals introduced into the mesocosms. At the end of the experiment, all fish were caught from each mesocosm and weighed (Kern ABT 220-4M). The four individuals were found in all mesocosms except for two replicates of the T treatment and one replicate of the TW treatment, where two, three and three fish were caught, respectively.