Impact of dust addition on the microbial food web under present and future 1 conditions of pH and temperature 2

21 In the oligotrophic waters of the Mediterranean Sea, during the stratification period, the 22 microbial loop relies on pulsed inputs of nutrients through atmospheric deposition of aerosols 23 from both natural (Saharan dust) and anthropogenic origins. While the influence of dust 24 deposition on microbial processes and community composition is still not fully constrained, the extent to which future environmental conditions will affect dust inputs and the microbial 26 response is not known. The impact of atmospheric wet dust deposition was studied both under 27 present and future (warming and acidification) environmental conditions through experiments in 28 300 L climate reactors. Three dust addition experiments were performed with surface seawater 29 collected from the Tyrrhenian Sea, Ionian Sea and Algerian basin in the Western Mediterranean 30 Sea during the PEACETIME cruise in May-June 2017. Top-down controls on bacteria, viral 31 processes and community, as well as microbial community structure (16S and 18S rDNA 32 amplicon sequencing) were followed over the 3-4 days experiments. Different microbial and 33 viral responses to dust were observed rapidly after addition and were most of the time higher 34 when combined to future environmental conditions. The input of nutrients and trace metals 35 changed the microbial ecosystem from bottom-up limited to a top-down controlled bacterial 36 community, likely from grazing and induced lysogeny. The composition of mixotrophic 37 microeukaryotes and phototrophic prokaryotes was also altered. Overall, these results suggest 38 that the effect of dust deposition on the microbial loop is dependent on the initial microbial 39 assemblage and metabolic state of the tested water, and that predicted warming, and acidification 40 will intensify these responses, affecting food web processes and biogeochemical cycles. 41 for min at °C. Virus particles were discriminated based on their green fluorescence Sea It was also shown to be repressed by dust addition in nutrient limited tropical Atlantic This suggests that microbial community in the tested waters. A different response in trophic interactions and

Input of essential nutrients and trace metals through aerosol deposition is crucial to the ocean 43 surface water biogeochemistry and productivity (at the global scale: e.g., Mahowald et al., 2017;44 in the Mediterranean Sea: e.g., Guieu and Ridame, 2020) with episodic fertilization events 45 driving microbial processes in oligotrophic regions such as the Pacific Ocean, the Southern 46 Ocean and the Mediterranean Sea.

47
The summer Mediterranean food web is characterized by low primary production (PP) and 48 heterotrophic prokaryotic production (more classically abbreviated as BP for bacterial 49 production) constrained by nutrient availability further limiting dissolved organic matter (DOM) 50 utilization and export, resulting in DOM accumulation. Therefore, inputs of bioavailable 51 nutrients through deposition of atmospheric particles are essential to this microbial ecosystem. 52 Indeed, these nutrient pulses have been shown to support microbial processes but the degree to 53 which the microbial food web is affected might be dependent on the degree of oligotrophy of the 54 water (Marín-Beltrán et al., 2019;Marañon et al., 2010). 55 In the Mediterranean Sea, dust deposition stimulates PP and N2 fixation (Guieu et al., 2014; Samples were taken at t-12h (while filling the tanks), t0 (just before dust addition), t1h, 104 t6h, t12h, t24h, t48h, t72h and t96h (after dust addition, and t96h only for FAST). 105 2.2. Growth rates, mortality, and top down controls 106 BP was estimated at all sampling points from rates of 3 H-Leucine incorporation 107 (Kirchman et al., 1985;Smith and Azam, 1992) as described in Gazeau et al. (2021). Briefly, 108 triplicate 1.5 mL samples and one blank were incubated in the dark for 1-2 h in two temperature-109 controlled incubators maintained respectively at ambient temperature for C1, C2, D1 and D2 and 110 at ambient temperature +3 °C for G1 and G2. HB, Synechococcus, picoeukaryotes and 111 heterotrohic nanoflagellates (HNF) abundances were measured by flow cytometry as described 112 in Gazeau et al. (2020). Bacterial cell specific growth rates were estimated assuming exponential 113 growth and a carbon to cell ration of 20 fg C cell -1 (Lee and Fuhrman, 1987). Net growth rates 114 (h -1 ) were calculated from the exponential phase of growth of BP, abundances of Synechococcus 115 and picoeukaryotes cells, observable from at least three successive sampling points. Mortality 116 was estimated as the difference between HB present between two successive sampling points and 117 those produced during that time. France) for 10 min at 80 °C. Virus particles were discriminated based on their green fluorescence and SSC during 1 min analyses (Fig. S1). All cytogram analyses were performed with the Flowing 126 Software freeware (Turku Center of Biotechnology, Finland).

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Viral production and bacterial losses due to phages were assessed by the virus reduction approach 128 (Weinbauer et al., 2010)  Falcon tubes. Three of the tubes were incubated as controls, while the other three were inoculated 135 with mitomycin C (Sigma-Aldrich, 1 µg mL -1 final concentration) as inducing agent of the lytic 136 cycle in lysogenic bacteria. All tubes were incubated in darkness in two temperature-controlled 137 incubators maintained respectively at ambient temperature for C1, C2, D1 and D2 and at ambient 138 temperature +3 °C for G1 and G2. Samples for HB and viral abundances were collected every 6 h 139 for a total incubation period of 18 h.

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The estimation of virus-mediated mortality of HB was performed according to Weinbauer et al. 141 (2002) and Winter et al. (2004). Briefly, increase in virus abundance in the control tubes represents 142 lytic viral production (VPL), and an increase in mitomycin C treatments represents total (VPT), 143 i.e., lytic plus lysogenic, viral production. The difference between VPT and VPL represents 144 lysogenic production (VPLG). The frequency of lytically infected cells (FLIC)  growth rates were also higher in D and especially in G relative to C (Table 2). Synechococcus

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and picoeukaryotes net growth rates showed a similar trend (Table 2). Heterotrophic bacterial 195 mortality was also higher than in C especially at TYR and in G at ION and FAST (Fig. 1). Over The abundance and production of virus-like particles (VLP) increased following an east 208 to west gradient (  ASVs related to SAR11 and Verrucomicrobia and Synechococcus decreased (Table S1a). At were assigned to an increase of ASVs related to different Alteromonas sp., Erythrobacter sp.,

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Verrucomicrobia, Rhodospirillales and some Flavobacteria ( to the difference between C and D/G (Table S1) while ASVs related to SAR11,

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Verrucomicrobia, Celeribacter sp. Thalassobius sp. and Rhodospirillales were mainly present in 267 C (Table S1c). Gymnodiniales and Gonyaulacales as well as to an increase in Chlorophyta (Table S2a). At ION, 277 no significant changes were observed between C and D/G after 24 h. However, after 72 h, the communities were significantly different in D (p = 0.018) and G (p = 0.05) compared to the 279 communities at t24h in these treatments (Table S2B). In D, diversity was significantly higher at 280 t72h compared to t24h and to C at the same sampling time (p = 0.036). In contrast, diversity in G 281 at t72h was lower than at t24h and lower to the one observed in C at the same sampling time (p = 282 0.066; Fig S6). These differences were mainly attributed to changes in ASVs related to 283 dinoflagellates and to the increase at t72h of Emiliana huxleyi and Chlorophyta in D and G, 284 respectively (Table S2b). At FAST, significant differences were observed between the controls 285 and initial communities compared to the dust amended (D and G) treatments at t24h (p = 0.036).

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No major differences were observed between D/G at t24h and t96h (p = 0.06). The differences 287 were mainly attributed to changes in dinoflagellates ASVs and to an increase in Acantharea and

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Emiliana huxleyi in D and G treatments at t96h (Table S2c).  following the east to west gradient of the initial water conditions.

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The dust addition induced similar nitrate + nitrite (NOx) and dissolved inorganic phosphate   , 2016;Westrich et al., 2016;Maki et al., 2016). Potential toxicity effects of metals released 441 from dust/aerosols on certain micro-organisms have also been reported (Paytan et al., 2009;442 Rahav et al., 2020). Here, the micro-eukaryotic community was dominated by a diverse group of 443 dinoflagellates which were responsible for the main variations between treatments at all stations.

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The overwhelming abundance of dinoflagellates sequences over other micro-eukaryotes could be biased by the large genomes and multiple ribosomal gene copies per genome found in 446 dinoflagellates (Zhu et al., 2005) or due to their preferential amplification. However, the  (Zhou et al., 2021). It was also shown to be repressed 466 by dust addition in nutrient limited tropical Atlantic (Marañon et al., 2010). This suggests that different Synechococcus ecotypes (Sohm et al., 2016) might respond differently to dust addition 468 depending on the initial biogeochemical conditions of the water.

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In the three experiments, the main bacterial ASVs responsible for the differences between    Microb, 70, 1506Microb, 70, -1513Microb, 70, , 10.1128Microb, 70, /aem.70.3.1506Microb, 70, -1513Microb, 70, .2004Microb, 70, , 2004.    between the treatments and the mean value of the duplicate controls. The first raw represents the 884 bacterial cell specific growth rates and relative mortality rates at t24h after dust addition. The second 885 raw represents the relative viral productions at t24h and at T0 for the G treatments. The last raw 886 represents the viral strategies: the percentages of lytic (FLIC) or lysogenic (FLC) cells at t24h and at T0 for 887 the G treatments. 888