Table 1.
Trypanosome infection outcomes in tsetse of differing age and symbiont status.
Table 2.
Trypanosome infection outcomes in tsetse of differing symbiont status.
Figure 1.
Immunity-related gene expression in teneral GmmWT flies following per os challenge with infectious trypanosomes.
Log2 fold-change in the expression of immunity-related genes in teneral GmmWT individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged teneral GmmWT individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log2 fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log2 fold-change for each gene assayed. All quantitative measurements were performed in duplicate. No significant difference in the expression of immunity-related genes was observed between challenged and unchallenged teneral GmmWT individuals at either of the monitored time points (Student's t-test).
Figure 2.
Immunity-related gene expression in mature GmmWT flies following per os challenge with infectious trypanosomes.
Log2 fold-change in the expression of immunity-related genes in mature GmmWT individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged mature GmmWT individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log2 fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log2 fold-change for each gene assayed. All quantitative measurements were performed in duplicate. Genes that presented a significant change in expression in parasite challenged versus unchallenged mature GmmWT flies are represented by red dots (p≤0.05; Student's t-test).
Figure 3.
Immunity-related gene expression in mature GmmApo flies following per os challenge with infectious trypanosomes.
Log2 fold-change in the expression of immunity-related genes in mature GmmApo individuals 24 hpc (A) and 3 dpc (B) with T. b. rhodesiense parasites. Gene expression in challenged and unchallenged mature GmmApo individuals is normalized relative to constitutively-expressed tsetse β-tubulin. All log2 fold-change values are represented as a fraction of average normalized gene expression levels in trypanosome-challenged vs. unchallenged flies. Samples sizes are represented by individual dots, and the red bars indicate the median log2 fold-change for each gene assayed. All quantitative measurements were performed in duplicate. Genes that presented a significant change in expression in parasite challenged versus unchallenged mature GmmApo flies are represented by red dots (p≤0.05; Student's t-test).
Figure 4.
Tsetse symbiont status correlates with structural integrity of the fly's peritrophic matrix.
(A) Midguts from 10 day old flies (3 days after consuming their last blood meal; n = 3) of each treatment group were microscopically dissected, fixed, sectioned and stained. Prepared sections were observed in an effort to compare PM structural integrity between tsetse treatment (GmmApo, GmmWT/Apo and GmmWT/Sgm−) and control (GmmWT) groups. Tsetse flies that underwent intrauterine larval developed in the presence of their endogenous microbiome (GmmWT, GmmWT/Apo and GmmWT/Sgm−) appear to have a structurally robust PM, while those that matured in the absence of their symbionts (GmmApo) do not. Red arrows identify the PM in gut sections where the structure was visible. 100× scale bars = 100 µm and 400× scale bars = 25 µm. (B) Dextran feeding assay of teneral GmmWT adults, and mature GmmWT and GmmApo adults (n = 10 per group). Flies were administered modified blood meals (see Materials and Methods, sub-section ‘Dextran feeding assay’, for details) supplemented with 500 kDa FITC-labeled dextran molecules. Six hours post-inoculation, midguts were dissected and examined under a fluorescence-emitting dissecting microscope. Scale bar (which is the same for all 3 panels) = 500 µm.
Figure 5.
Age and symbiont status modulate trypanosome infection outcomes in the tsetse fly.
Approximately 50% of teneral WT tsetse flies become infected when challenged with trypanosomes. Flies at this stage of development exhibit an immature PM, and present a weak and innocuous innate immune response following parasite challenge. Some teneral tsetse flies are refractory to parasite infections, likely because they acquire more maternally-transmitted PGRP-LB than their susceptible counterparts. Mature GmmWT flies present a vigorous immune response following challenge with trypanosomes and are thus highly resistant to parasite infection. In contrast, age-matched GmmApo flies, which undergo their entire lifecycle (including intrauterine larval development) in the absence of endogenous microbes, are relatively susceptible to trypanosome infection. Although mature GmmApo flies also up-regulate the expression of several immunity-related genes following trypanosome challenge, notably absent from this list is trypanolytic pgrp-lb. Interestingly, the timing of this response also occurs earlier in the infection process in GmmApo compared to GmmWT individuals. We propose that this premature immune response results from the fact that aposymbiotic tsetse house a structurally compromised PM that allows these flies to detect parasites immediately upon entry into the fly's midgut. Our model suggests that symbiotic microbes present in larval tsetse modulate the ability of subsequent adults to produce an intact PM. In turn, this structure regulates trypanosome infection outcomes by controlling the timing of tsetse's immune response following parasite challenge. Other invertebrates, including D. melanogaster and Hirudo verbana (the medicinal leech), house phagocytic cells in their alimentary canal that engulf pathogenic organisms [55], [56]. We speculate that WT tsetse may house similar cells in it's digestive tract that assist in the fly's immune response against trypanosome challenge. PV, proventriculus; BT, bacteriome; GL, gut lumen; EPS, ectoperitrophic space; CP, crop; PM; peritrophic matrix; AMP, antimicrobial peptide; ROS, reactive oxygen species; PGRP-LB, peptidoglycan recognition protein LB.