Discovery of a Natural Microsporidian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans
(a) The anterior region of a C. elegans animal co-infected with N. displodere (green) and N. parisii (red), visualized by FISH using species-specific rRNA probes and DAPI (blue). This image was captured by confocal microscopy with a single z-plane represented in the main inset, and orthogonal views of the x- and y-planes on the top and right insets, respectively, which show a cross-sectional view of the captured z-stacks within those planes. Scale bar is 50 μm. (b) C. elegans tissue-specific GFP-expression strains in the epidermis (top), body wall muscle (middle), and neurons (bottom), were infected with N. displodere and imaged at 3 dpi by FISH and DAPI. The neuron infected was in the ventral nerve cord (bottom). (c) Tissue-specific GFP strains were infected and imaged at 5 dpi with FISH and DY96 to stain clusters of spores (Sp). GFP-positive tissues that are difficult to see due to heavy infection are outlined with dashed white lines. The neuron infected was in the pre-anal ganglia (bottom). Scale bars are 20 μm. (d) The mid-body of the C. elegans intestinal GFP-expression strain infected with N. displodere at 1 dpi (top) and 3 dpi (bottom). Infection events are labeled as either inside (in) or outside (out) of the GFP-labeled intestine. Scale bar is 10 μm. (e) The tissue distribution of proliferating N. displodere infection was analyzed at 3 dpi, and was calculated individually in each C. elegans tissue-expression strain as the percent of FISH-stained meront clusters occurring in the GFP-positive tissues compared to the total number of events throughout the animals. Data are represented as the mean with SD of four replicates across two experiments, with a total of 50 animals counted for each replicate. (f) A comparison of the percent of animals infected in the specified GFP-positive tissue at three time points at which the three main stages of N. displodere infection occur, with sporoplasms analyzed at 1 dpi, meronts at 3 dpi, and new spores at 6 dpi. Each time point was calculated individually in each C. elegans tissue-expression strain as the percent of 50 animals that show a given symptom in the GFP-positive tissues. Data are represented as the mean with SD of four replicates across two experiments (ns = not significant, comparing intestine to muscle (p = 0.55) or intestine to epidermis (p = 0.11) at 1 dpi; *p = 0.03 comparing intestine to muscle and comparing intestine to epidermis at 6 dpi, two-tailed Mann-Whitney test).