17 The presence of pico-sized cyanobacteria (genus Synechococcus) in hypolimnetic waters 18 has been reported, and investigators have suggested that Synechococcus make a greater 19 contribution to ecological processes in the hypolimnion than previously hypothesized. 20 However, the ecological role of Synechococcus in food webs and/or matter cycling in the 21 hypolimnion remains unknown. To address this issue, we assessed protistan grazing and the 22 Synechococcus:bacteria carbon biomass and

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... y grazing loss ratios were 10.8 and 11.0%, 30 respectively. Thus, it is likely that the Synechococcus biomass is an important seasonal 31 component of the carbon flux in the hypolimnetic microbial loop. Our results provide the 32 first data on carbon flux through the mortality of both Synechococcus and bacteria in a 33 hypolimnetic ecosystem. 42 The widely distributed cyanobacterial genus Synechococcus is a major component 43 of photosynthetic biomass in freshwater lakes (Sigee 2005). Synechococcus, which are 44 among the smallest prokaryotes in phytoplankton communities, are vulnerable to 45 microzooplankton grazing. Previous studies have suggested that most of their production is 46 rapidly removed from the euphotic zone (Nagata 1988). Conversely, other researchers have 47 reported the presence of pico-sized cyanobacteria in hypolimnetic waters (Callieri and 48 Pinolini 1995, Takasu et al. 2015). Previously, we revealed that substantial numbers of 49 intact Synechococcus cells were retained among larger organic particles that had sunk to the 50 hypolimnion (Takasu et al. 2015). Thus, Synechococcus might be an important food source 51 and/or item for hypolimnetic grazers. However, information about the fate of 52 Synechococcus in a hypolimnetic ecosystem is limited, and the role of Synechococcus in 53 food web and/or matter cycling remains unknown. 54 Protistan grazing and viral lysis are two important determinants of the fate of 55 Synechococcus and heterotrophic bacteria (Sigee 2005). Protistan grazing transfers the 56 prokaryotic biomass to organisms at higher trophic levels via the microbial loop, whereas 57 viral lysis leads to the recycling of carbon and nutrients, each of which is derived from the 58 lysed prokaryotic biomass and re-supplied to prokaryotes (Sigee 2005). Thus, it is 59 important to characterize the relative contributions of grazing and lysis to Synechococcus 60 mortality in order to understand their role in ecological processes in the hypolimnion. 61 In the present study, we hypothesized that Synechococcus contributes to the food 62 web and/or matter cycling in the oxygenated hypolimnion of Lake Biwa. To investigate this 63 hypothesis, we assessed protistan grazing and virus-mediated Synechococcus mortality. To 64 evaluate the role of Synechococcus within the hypolimnetic ecosystem, we compared the 65 carbon flux through Synechococcus mortality to that of bacteria. 66 67 Study site 68 Lake Biwa is a large (surface area, 674 km 2 ; water volume, 27.3 km 3 ; watershed 69 area, 3848 km 2 ), deep (maximum depth, 104 m), tectonic, freshwater (average 70 concentrations of Cl, Na and Ca are 7.5, 5.2 and 10.4 mg L -1 、respectively; Fujinaga et al. 71 2005) lake in Japan. The mesotrophic and monomictic north basin of the lake has a water 72 residence time of 5.5 years. We collected water samples at station Ie-1 (35° 12' 58" N, 135° 73 59' 55" E; ca. 75 m) in the north basin of the lake. The water column is vertically mixed 74 from January to March and stratified during the rest of the year. 75 76 Methods 77 the stratification period. Vertical profiles of water temperature and light intensity were 79 determined using a CTD probe (SBE 911 Plus; Sea Bird Electronics, Bellevue, WA, USA). 80 Water samples were collected using Niskin X bottles. 81 To determine chlorophyll a (chl a) concentrations, water samples of 285 ml 82 collected at depths of 5 m (epilimnion) and 65 m (hypolimnion) were filtered through 0.2-83 and 2.0-µm polycarbonate filters (Whatman International, Ltd., Maidstone, England) and 84 analyzed by fluorometry (Fluorometer 10-AU; Turner Designs, Sunnyvale, CA, USA) 85 according to Welschmeyer (1994). Chl a concentrations in the 0.2-2.0-µm fraction 86 104 nanoflagellates (HNF), and 0.1 mL (1 mL from samples diluted 10 9 with 0.02-μm-filtered 105 distilled water) was used for the enumeration of viral-like particles (VLP). HNF were 106 double-stained with DAPI (final concentration: 10 μg mL -1 ) and fluorescein isothiocyanate 107 (Dojindo Molecular Technology, Inc., Rockville, MD, USA; final concentration: 10 μg 108 mL -1 ) for 10 min, collected on 0.8-μm-pore-size black polycarbonate filters (Whatman), 109 and counted using epifluorescence microscopy under ultraviolet (UV; U-MWU2; Olympus) 110 and blue (IB-NIB; Olympus) excitation according to Sherr and Sherr (1983). For HNF 111 counting, a minimum of 100 randomly selected fields were inspected. VLP were counted 112 314 Apple JK, Strom SL, Palenik B, Brahamsha B. 2011. Variability in protist grazing and 315 growth on different marine Synechococcus Isolates. Appl. Environ. Microbiol. 316 77:3074-3084. 317 Callieri C, Pinolini ML. 1995. Picoplankton in Lake Maggiore, Italy. Int. Revue. ges. 318 Hydrobiol. 80:491-501. 319 Caron DA, Lim EL, Miceli Geraldine, Waterbury JB, Frederica WV. 1991. Grazing and 320 utilization of chroococcoid cyanobacteria and heterotrophic bacteria by protozoa in 321 laboratory cultures and a coastal plankton community. Aquat. Microb. Ecol.