Fig. 2: Extracellular vesicles modulate viral infection of E. huxleyi and prolong the half-life of EhV in natural assemblages.

Vesicles derived from lab cultures of E. huxleyi CCMP2090 were incubated with natural microbial populations from (a) the early bloom phase (day 14, blue arrow in Fig. 1a) or (b) demise phase (day 20, red arrow in Fig. 1a). Abundance of nanophytoplankton (including calcified and non-calcified E. huxleyi), calcified E. huxleyi, EhV-like particles, Synechococcus, and bacteria was measured by flow cytometry 48 h post-treatment. In (a), significant difference between the treated and untreated populations was observed only for bacteria (***p value = 0.001). For untreated n = 10, for vesicle-treated n = 30. In (b), significant reduction in nanophytoplankton abundance (***p value = 0.0003), concomitant with elevation in EhV-like particle count (**p value = 0.009) and Synechococcus abundance (***p value = 1.03 × 10⁻⁷) was observed. For both treated and untreated n = 30. At least 10,000 events were counted for each gate. In (a) and (b), p value was calculated using two tailed t test with equal variance. c Half-life of infectious EhV from the mesocosm bags, measured by the most probable number (MPN) method. EhV was sampled from the bags during the demise phase of the bloom (day 18, green cross in Fig. 1a). Vesicles from infected (^VirusVesicles) or uninfected (^ControlVesicles) lab cultures of E. huxleyi CCMP2090 were added to natural EhV at a ratio of 10 vesicles per EhV-like particle. For the ^Controlvesicle treatment, the decay was so fast we could not detect infectivity in more than one time point. Therefore, the minimum detectable infectivity values were used in the subsequent time points in order to calculate the maximum possible half-life. Average ± SE is presented. ***p value < 0.001 for each treatment compared to the untreated control, using ANOVA with Dunnett’s post-hoc test, n = 3.