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    Livestock enclosures in drylands of Sub-Saharan Africa are overlooked hotspots of N2O emissions

    Field N2O flux measurements
    At seven different semi-arid and arid savanna regions in Kenya (Fig. 1, Supplementary Fig. 9), N2O measurements from soils at 46 boma sites and 22 adjacent reference sites (undisturbed savanna) were carried out. At each boma and control site, 3–7 plots were chosen randomly for flux measurements. Local members of the pastoral communities, including herders and/or community elders were interviewed for information on the time since abandonment of each boma.
    N2O fluxes from bomas were measured using the fast-box chamber method15, deploying an ultra-portable greenhouse gas analyzer of ABB-Los Gatos Research Inc. (Modell 909–0041). A gas-tight, vented chamber (0.3 × 0.2 × 0.15 m) was pressed against the ground on foam frames for 4–7 min, during which time sample air was pumped from the headspace of the chamber to the analyzer and returned to the chamber thereafter. In this way, changes in headspace N2O concentrations were continuously measured over the sample period, with a running average of every 5 s. Linear regression over the sample period was used to calculate fluxes. The detection limit for N2O fluxes was More

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    Temperate infection in a virus–host system previously known for virulent dynamics

    Host cultures
    All laboratory experiments were conducted with Emiliania huxleyi strain CCMP374 (https://ncma.bigelow.org/ccmp374) and EhV strain 207 (see below). CCMP374 is a naked strain of E. huxleyi isolated from the Gulf of Maine in 1990, and exhibits rapid growth, high-stationary phase densities (~107 cells per milliliter), and high sensitivity to viral infection27,63. Cultures were maintained at 5·105 to 1·106 cells per millilier and grown at 18 °C, with a 14 h:10 h light:dark cycle with a light intensity of 125 µmol photons m−2 s−1. CCMP374 was grown in batch culture conditions with f/2 rich nutrients42 added to 0.2 µm pore-size filtered (GE Healthcare USA, filter 6718-9582) autoclaved seawater in either polystyrene 50 mL flasks or 6-well plates or polypropylene 96-well plates (Greiner Bio-One, USA; items 690160, 657185, and 780270, respectively; Supplementary Table 1). Addition of f/2 nutrients increases macronutrient concentrations (e.g., NaNO3 882 µM; an ~88-fold enrichment over basal seawater with ~10 µM NaNO3; other nutrients see similar enrichments). This provides ideal, replete conditions conducive to virulent dynamics in which to probe for the presence of virulent viral behavior.
    Virus cultures
    EhV207 has commonly been used to elucidate virulent dynamics, as it induces the rapid decline of host populations and concomitant production of high titers of viral progeny under culture conditions28,36,64. Together with CCMP374, EhV207 comprises a highly virulent host–virus system, strongly predisposing this work towards the execution of virulent activity. Viruses were cultured by adding them to exponentially growing cultures at ~5·105 to 1·106 cells per milliliter in f/2 media at a virus:host ratio of 10:1 MOI. Cultures visibly cleared after approximately three days and viruses were isolated from cellular debris using 0.45 µm pore-size filtration (EMD Millipore, USA; filters SLHV033RS or SVHV01015) and lysates stored in the dark at 4 °C until use within 1 week. This approach yielded viral titers in excess of 108 viruses per milliliter. In experiments where a virus-negative control was required, a heat-killed lysate was produced by incubation at 90 °C for 10–20 min prior to 0.02 µm pore-size filtration (Anotop, Whatman, USA) and cooling to ~18 °C. All infections were conducted in the morning41. For all experiments, virus infectivity was monitored by running parallel cultures with initial host densities of 105 cells per milliliter coincubated with a MOI of 10 (10:1 viruses:host). These visibly cleared in all cases, showing that our viruses were always infectious in these experiments. All flasks were shaken daily and plates mixed by pipetting to preclude settling and ensure equal exposure to infection. In summary, all experiments were conducted in a manner typically conducive to virulent infection and with viable viruses and sensitive hosts.
    Laboratory coincubation experiments
    E. huxleyi-EhV virulent infection dynamics were first were studied using coincubation of viruses and hosts at an initial ratio of 10:1 viruses:host (MOI = 10) in laboratory conditions. In experiments without preinfection treatments—Experiments II, III, and VII—viral lysates were added to high-density (~1·106 cells per milliliter; quantified using a Coulter Counter Multi-sizer 3, Beckman, USA) cultures to a final ratio of 10:1 virus:host (MOI = 10; viruses were quantified using an Influx Mariner flow cytometer; BD, USA). Cultures were then serially diluted down to experimental densities; all cells were from the same inoculum within each experiment (see Supplementary Table 1 for densities in each experiment and Supplementary Fig. 3 for experimental rationale). Uninfected controls substituted lysates with heat-killed, filtered lysate. In experiments with preincubation treatments (Experiments I, IV, V, and VI), cultures for 10:1 MOI coincubation treatment were drawn from the uninfected control after centrifugation and washing, so that uninfected controls, 10:1 MOI coincubation, and pre-infected treatments were all subjected to similar centrifugation and washing before lysate/heat-killed viral addition. The initial set of experiments (Experiments I, II, III, and VII) were conducted for approximately a week as we expected lysis to occur at all densities in that time (Supplementary Table 1). These experiments were subsequently repeated due to our initial interpretation that the lack of lysis in scarce densities ( More