Figure 1.

The circulating IgM⁺ B cell compartment of the common carp expands upon infection with the myxozoan S. molnari or is depleted upon immunosuppression. (A) A representative gating strategy for quantifying the IgM⁺ B cells in the whole blood of common carp. From the leftmost to the rightmost plots, dashed arrows each point out gated subpopulations (labeled with their proportions) being further analyzed in the plot to their right. The oval morphology of fish red blood cells gives them a heterogeneous scatter profile that overlaps with that of leukocytes. To exclude them, we first broadly gated on cells with low forward scatter width (FSC-W, x-axis), representing all leukocytes. Subsequently, doublets were excluded based on disproportionate side scatter (SSC) width (W) and height (H). Cells stained with the mitochondria- and endoplasmic reticulum-specific DiOC6 (y-axis) can distinguish active cells (leukocytes) from relatively inactive cells (red blood cells) with lower mitochondria or endoplasmic reticulum content. Among the leukocytes, the FSC^low SSC^low population represents lymphocytes which include IgM⁺ B cells based on staining with the monoclonal anti-carp IgM antibody WCI12 (mean fluorescence intensity, y-axis of the rightmost plot). Through this strategy, (B) we quantified the number of IgM⁺ B cells (gate and proportions indicated) throughout the experiment: from an early timepoint (day 3, top row) to a late timepoint (day 41, bottom row). Each column represents separate experimental groups either infected with 2,500,000 parasites (S. molnari), immunosuppressed and infected with an inoculum of 2,500,000 parasites (IS + S. molnari), or mock-infected (Mock). Representative plots are shown.