Tsetse Immune System Maturation Requires the Presence of Obligate Symbionts in Larvae
Figure 3
Hemocyte-mediated phagocytosis is a critical component of tsetse's immune response.
(A) 8-d-old GmmWT were subjected to septic infection with GFP-expressing E. coli K12. Twelve hours post-infection hemolymph was collected, fixed on glass slides using 2% paraformaldehyde, and microscopically examined for the presence of hemocyte-engulfed bacterial cells. Scale bar = 10 µm. (B) The process of hemocyte-mediated phagocytosis in tsetse was blocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WT individuals. In consecutive 12 h intervals following bead injection, flies were infected with GFP-expressing E. coli K12 and then hemolymph was collected and fixed as described above. Hemocytes appear to have engulfed the beads, thus prohibiting the subsequent uptake of bacterial cells. The inset in each panel shows a higher magnification image of one hemocyte, which is identified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytes incapable of engulfing E. coli are susceptible to septic infection with this bacterium while their wild-type counterparts are not. The susceptible phenotype is exhibited regardless of whether phagocytosis-inhibited tsetse were inoculated with 103 or 106 CFU of E. coli. Beads alone had no effect on tsetse mortality. No significant difference existed in survival outcome between mature GmmWT phagocytosis inhibited flies infected with 103 versus 106 CFU of E. coli (p = 0.47, log-rank analysis). Furthermore, no significant difference was present between mature GmmWgm− flies with uninhibited hemocytes (Figure 1A, bottom panel) and mature GmmWT phagocytosis inhibited flies (p = 0.11) infected with 106 CFU of E. coli.