Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection

Abstract

A novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection was investigated in Anopheles stephensi. The presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway and is proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Silencing components of key signaling pathways through RNA interference (RNAi) that enhance proliferation of progenitor cells significantly decreased oocyst numbers, while limiting proliferation of progenitors increased oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts.

Fig. 1. Response of Anopheles stephensi HP10 line midgut progenitors to Plasmodium berghei infection.

A Small triangular tdTomato⁺ cell in the basal side of the midgut epithelium. B Larger tdTomato⁺ cells embedded in the epithelium. Front and lateral views. Scale bar: 7 and 5 µm, respectively. td-Tomato (red), nuclei (blue) and actin (green). L= lumen and B= basal. C Delta antibody staining of small triangular tdTomato⁺ cells. D Close-up of delta immunostaining in small triangular cells. tdTomato in red; Nuclei in blue and delta in green. Scale bars: 7 µm. Micrographs are representative of experiments that were independently reproduced at least 2 times with multiple individuals each experimental group obtained similar results. tdTomato⁺ cells in midguts (E) 24 h, (F) 5 days and (G) 10 days PI. Parasites (green), tdTomato (red) and nuclei (blue). Volume of tdTomato (tdTomato⁺ cells) in the mosquito midgut at (H) 24 h (p = 0.9770) (I) 5 days (p < 0.0001) and (J) 10 days PI (p < 0.0001). Each dot represents the volume of red fluorescence for individual midguts and the medians are indicated with the horizontal line. Two-tailed Mann Whitney U test, ns = p > 0.05. Percentage of Plasmodium parasites that are in contact with midgut progenitors at (K) 24 h, (L) 5 days and (M) 10 days PI. C = uninfected midguts, I = infected midguts. n = number of parasites. The numerical data underlying the plots in the manuscript are provided as a Source Data File.

Fig. 2. Effect of Plasmodium infection on midgut architecture and of oocysts intensity of infection on proliferation of midgut progenitors.

A Actin staining of blood-fed control midguts. B Actin staining in P. berghei-infected midguts 10 days PI. Oocysts in the epithelia are indicated by yellow asterisks. C Actin staining (cyan), progenitors (red) and P.berghei oocysts (green). Scale bar: 30 µm. D, E Progenitor “ribbons” (red) surrounding oocysts (green), 10 days PI. F Actin (cyan) staining showing close association of progenitors (red) embedded in the epithelium with oocysts (green), 10 days PI. Scale bar: 10 µm. G Lateral view of (F), showing midgut progenitors (red) surrounding an oocyst 10 days PI. L = lumen and B = Basal. Micrographs are representative of experiments that were independently reproduced at least 2 times with multiple individuals in each experimental group obtained similar results. H Midgut progenitors and oocysts in the midgut of An. stephensi kept at (H) 21 °C and (I) 28 °C. J Oocysts counts of mosquitoes kept at 21 °C, a permissive temperature for oocyst development, and at 28 °C, a non-permissive one. Each dot represents the number of oocysts on individual midguts. The median is indicated by the horizontal line. Two-tailed Mann-Whitney U test (p < 0.0001). K Volume of tdTomato (tdTomato⁺ cells) 10 days PI of females kept at 21 °C and 28 °C. Each dot represents the volume of red fluorescence for individual midguts and the medians are indicated by the horizontal line. Two-tailed Mann Whitney U test (p < 0.0001). L Correlation of oocyst numbers and tdTomato volume (tdTomato⁺ cells) of midguts from females kept at 21 °C (red dots) and 28 °C (gray dots). Each dot represents an individual mosquito. Linear regression, r² = 0.9019, p < 0.0001. The numerical data underlying the plots in the manuscript are provided as a Source Data File.

Fig. 3. Effect of proliferation of midgut progenitors on oocysts survival.

A LacZ and (B) SOCS-silenced midguts 10 days PI. C Volume of tdTomato (tdTomato+ cells) in lacZ and SOCS-silenced midguts 10 days PI (p = 0.0013). D Oocyst counts of lacZ, and SOCS silenced midguts 10 days PI (p = 0.0004). E lacZ and (F) HOP-silenced midguts 10 days PI. G Volume of tdTomato (tdTomato+ cells) in lacZ and HOP-silenced midguts 10 days PI (p < 0.0001). H Oocyst counts of lacZ and HOP-silenced midguts 10 days PI. I LacZ and (J) delta-silenced midguts 10 days PI (p = 0.009). K Volume of tdTomato (tdTomato+ cells) in lacZ and delta-silenced midguts 10 days PI (p = 0.0003). L Oocyst counts of lacZ and delta-silenced midguts 10 days PI (p = 0.0004). Each dot represents an individual mosquito. Median is indicated by the horizontal line. Two-tailed Mann Whitney U test. Midgut progenitors are shown in red and P. berghei oocysts in green. Scale bars: 40 µm. The numerical data underlying the plots in the manuscript are provided as a Source Data File. Micrographs are representative of experiments that were independently reproduced at least 2 times with multiple individuals in each experimental group and obtained similar results.

Fig. 4. Midgut progenitors actively eliminates Plasmodium oocysts and internalize parasite remnants.

A Whole mosquito live imaging over the course of 9 h 10 days pi. Scale bar from top view: 20 µm and from side view: 30 µm. B Whole mosquito live imaging over the course of 12 h 14 days PI. Scale bar from top view: 20 µm and from side view: 50 µm. C Oocyst fragments inside midgut progenitors at 5 days PI. White arrows indicate parasite remnants inside of midgut progenitors. Scale Bar: 15 µm. Leftover of P.berghei GFP from inside of a midgut progenitor 5 days PI (D) Front view and (E) Lateral view. Scale Bar: 5 µm and 7 µm respectively. Midgut progenitors are in red and P.berghei are in green (F) Dead oocyst in cyan inside of a midgut progenitor 5 days PI. Scale Bar: 10 µm. G Dead oocysts in cyan inside midgut progenitors 5 days PI. Scale Bar: 10 µm. H Live oocysts with cyan staining on the surface and a smaller dead oocyst with staining inside the capsule, engulfed by a midgut progenitor. White arrows indicate dead oocysts. Scale Bar: 15 µm. Micrographs are representative of experiments that were independently reproduced at least 2 times with multiple individuals in each experimental group and obtained similar results.