Fig 1.
Crithidia fasciculata adherent stages in culture and the mosquito host.
(A) Phase contrast images of live swimming (top panel) and adherent rosettes (bottom panel) grown in culture. Scale bars, 10 μm. (B) Images of Crithidia rosettes within an infected Aedes aegypti hindgut. Top panel shows an infected hindgut. C. fasciculata colonize the hindgut, rectum and rectal papillae (RP). Scale bar, 100 μm. Bottom panels are enlargements of the areas shown in the boxes above. Scale bars, 20 μm. Adherent cells are visible throughout the highlighted regions. White arrowheads in bottom panels indicate rosettes (C) Growth curve comparing rates of growth of cells grown in flasks kept on a rocker (shaking, solid line) and the incubator shelf (stationary, dotted line). Images of cells in shaking and stationary flasks. White arrowhead indicates a rosette. Scale bar, 25 μm. (D) Top image, cells that were allowed to adhere for 2 h followed by 3 washes to remove swimming cells. Bottom image shows the same flask after adherent cells have been allowed to grow for ~24 h. Scale bar, 25 μm.
Fig 2.
Organization of cultured C. fasciculata rosettes.
(A) Live C. fasciculata swimming cells expressing cytoplasmic GFP from an episomal plasmid. Scale bar, 10 μm. (B) Live GFP-expressing rosettes grown on poly-L-lysine-coated glass coverslips. Two different focal planes are shown in phase contrast to highlight the three-dimensional structure of the rosette. Scale bar, 10 μm. (C) Enlargement of rosette cells expressing GFP. Arrowhead indicates possible junction between cells. Scale bar, 10 μm.
Fig 3.
Time course Differential Interference Contrast live microscopy of cells in a rosette.
(A) Panels are representative frames taken over 20 h of a 24 h imaging period. During this time, several cell divisions were observed. Times are shown as h:min:s. (B) Cells imaged live over approximately 4 h. Black arrowheads indicate cells in the process of division, while open arrowheads indicate divided cells about to leave the rosette. Times are shown as h:min.
Fig 4.
Hindgut of Ae. aegypti mosquito 7 days post infection with GFP-expressing C. fasciculata.
(A) Widefield fluorescence microscopy was used to image fluorescent parasites in a dissected mosquito hindgut. Parasites are observed attached to the hindgut, rectum and rectal papillae. (B) Higher magnification image of the bracketed region indicated in panel A. The scale bars in panels A and B are 100 μm and 20 μm, respectively.
Fig 5.
RNAseq analysis of swimming and adherent C. fasciculata.
(A) The number of reads mapped to the C. fasciculata genome from cultured swimming, cultured adherent (left Y-axis), and infected mosquito hindgut samples (right Y-axis). Very few reads mapped in our mosquito samples, but subsampling indicates that our results are reproducible and not the result of differences in read depth. (B) Analysis of adherent and swimming C. fasciculata generated in vitro. Principle component analysis (PCA) shows a clear separation between cultured swimming and cultured adherent sequenced samples in PC1, indicating that adherence accounts for most of the variability between these samples. (C) Volcano plot showing transcript abundance in swimming vs. adherent cells (positive value indicates transcript is up in adherent cells). Dots shown in red represent transcripts with a log2 fold change ≥1 and an adjusted P value p<0.01. Numbers of differentially regulated genes are shown in the graph inset. (D) PCA of all samples including C. fasciculata reads from infected mosquito hindguts. (E) Volcano plot showing abundance of C. fasciculata transcripts in mosquito hindgut samples versus cultured swimming or (F) cultured adherent samples. Positive value indicates transcript is up in hindgut samples. Red dots represent transcripts with a log2 fold change ≥1 and an adjusted P value p<0.01. Numbers of differentially regulated genes are shown in graph insets.
Fig 6.
Differentially expressed genes in cultured swimming and cultured adherent C. fasciculata.
(A) Heat-map showing gene expression levels of genes differentially expressed by at least two-fold with an adjusted P value p<0.01 across three replicates of swimming cells and three replicates of adherent cells. Replicate number is shown at the top of the heat map. Each row represents a gene, and rows were clustered according to their expression pattern. Group 1 genes are upregulated in swimming cells relative to adherent cells. Group 2 genes are downregulated in swimming cells relative to adherent cells. Red, upregulated; green, downregulated. (B)(C) Gene ontology (GO) enrichment analysis (using GO Slim terms) of genes in groups shown in A was performed on TriTrypDB. p<0.05. (D) Analysis of differentially expressed genes in all nine samples (three replicates each of swimming, adherent, and infected hindguts). Venn diagram showing the degree of overlap in transcripts upregulated in cultured adherent parasites and hindgut-adherent parasites (both relative to cultured swimming cells). GO Slim terms enriched in each group (p<0.05) are shown in color-coded graphs on the right.
Fig 7.
gp63 genes are differentially expressed in adherent versus swimming C. fasciculata.
Heat map showing gene expression patterns of predicted gp63 genes in the C. fasciculata Cf-C1 genome across our different samples. The five genes outlined by the yellow box were significantly enriched in our cultured adherent samples by at least two-fold with an adjusted P value p<0.01.
Table 1.
Gene sets enriched in adherent and swimming form C. fasciculata.
Fig 8.
GSEA of putative paraflagellar rod proteins.
The left panel shows a heat map of enrichment in adherent (Adh) or swimming (Swim) forms for the three individual replicates (bottom of image). Gene identifiers and TriTrypDB gene name are shown. Red color indicates transcript enrichment in swimming form and blue indicates enrichment in adherent form. The 25 genes below the red dashed line (GSEA software threshold) are considered as significantly contributing to the enrichment of the set. The right panel shows the enrichment plot generated by the GSEA software with the Normalized Enrichment Score (NES) and False Discovery Rate (FDR) indicated for this gene set. A negative NES indicates enrichment in swimmers. The gray shaded area shows a rank order of the genes based on their enrichment in adherent (left, red) or swimming forms (right, blue). The black vertical lines indicate the position of the 46 members of the set within the rank order. The density of lines on the right indicates the strong enrichment of genes in swimming form. The green line indicates the enrichment profile generated by the software.
Fig 9.
Differentially regulated genes in C. fasciculata-infected Ae. aegypti hindguts.
(A) PCA analysis shows that before batch correction (left) the most significant source of variation is experimental replicate (Rep; different colors). Applying batch correction (right) separates control, uninfected (open circles) from infected samples (solid circles) along PC1, which now accounts for 47% of the variation between samples. (B) Volcano plot showing changes in gene expression in infected hindguts compared to uninfected hindguts. A positive value indicates an Ae. aegypti transcript that is upregulated in infected hindguts. Red dots indicate transcripts that are significantly differentially expressed. Numbers of differentially expressed genes with a log2 fold change of at least 0.59 and adjusted P value < 0.05 are shown in the inset.