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[GUIDELINES]
<strong>Note:</strong> It is recommended that rates of bacterial production be measured simultaneous to all time points for virus measurements. There are two reasons for this: (1) if measured simultaneously, the researcher can estimate virus production throughout the experiment, rather than relying simply on bacterial production estimates from time zero; and (2) bottle effects are common in small-volume experiments such as these. Measurements of bacterial production throughout the experiment will help the researcher to identify times when bacterial production is heightened (or reduced) due to bottle effects.<br /><br /><strong>Calculation of virus production and removal rates<br /><br /></strong> Production and removal rates are calculated from the equations of Glibert et al. (1982) and Fuhrman (1987). The decay constant, k, is calculated as<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bb9c2xe.png" alt="" width="99" height="68" data-src="https://s3.amazonaws.com/pr-journal/bb9c2xe.png" data-ofn="FLV tracer eq 1.png" /><br />where t is the incubation time and R<sub>0</sub> and R<sub>t</sub> are the ratios of labeled to unlabeled viruses at time 0 and time t, respectively. The first two time points in this experiment were t<sub>0</sub> and t<sub>1</sub>. For example, R<sub>0</sub> is FLV<sub>0</sub>, divided by the number of total number of virus particles (stained and unstained), C<sub>0</sub>, at time 0.<br /><br />The mean specific activity,<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bb5c2xe.png" alt="" width="22" height="22" data-src="https://s3.amazonaws.com/pr-journal/bb5c2xe.png" data-ofn="FLV tracer r symbol.png" /> , is then calculated as<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bcac2xe.png" alt="" width="216" height="47" data-src="https://s3.amazonaws.com/pr-journal/bcac2xe.png" data-ofn="FLV tracer eq 2.png" />    <br /><br /><br />The viral decay or removal rate, D<sub>v</sub> , is calculated as <br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bb8c2xe.png" alt="" width="183" height="59" data-src="https://s3.amazonaws.com/pr-journal/bb8c2xe.png" data-ofn="FLV tracer eq 3.png" /><br />where FLV<sub>0</sub> and FLV<sub>t</sub> are the concentrations of FLV at t<sub>0</sub> and at time t, respectively.<br /><br /><br />The viral production rate, P<sub>v</sub> , is calculated as<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bcbc2xe.png" alt="" width="238" height="97" data-src="https://s3.amazonaws.com/pr-journal/bcbc2xe.png" data-ofn="FLV tracer eq 4.png" /><br />where C<sub>0</sub> and C<sub>t</sub> are the concentrations of virus particles at t<sub>0</sub> and time t, respectively. <br /><br />If the virus abundance does not change over time, then the removal rate is equal to the production rate (and the equation is not used). For each experiment, initial rates (using the first two time points, t<sub>0</sub> and t<sub>1</sub>) and overall rates (using the entire time course) of production and decay are calculated. Initial rates of decay/production are closest to in situ rates, as all of the experiments can be started at dusk and held under ambient natural conditions. Overall rates represent decay/production under natural conditions for ~12 h, but samples held in the dark the following morning should not be exposed to natural sunlight.<br /><br />Estimates of viral-induced bacterial mortality can be calculated using overall rates of virus production, mean viral abundance, mean bacterial abundance and growth rates, and either an empirically measured or estimated burst size. Briefly, virus production rates are divided by the estimated burst size (we used a range from 20 to 50) to determine the number of bacterial cells killed L<sup>–1</sup> day<sup>–1</sup>. The researcher can divide the number of bacterial cells killed L<sup>–1</sup> day<sup>–1</sup> by the rate of bacterial production in cells L<sup>–1</sup> day<sup>–1</sup>, to determine the portion of the bacterial community killed due to viral lysis. All of the provided calculations assume steady state.<br /><br /><br />

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