Metabolic signatures of triatomine vectors of Trypanosoma cruzi unveiled by metabolomics

Abstract

Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which T. cruzi develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (Rhodnius prolixus, Triatoma infestans, Panstrongylus megistus) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect T. cruzi development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific T. cruzi strains. Knowledge of the chemical environment of T. cruzi in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.

Figure 1. Distribution of metabolites in triatomine feces.

A: Negative ions, B: Positive ions, C: Sum of negative and positive ions.

Figure 2. Distribution of metabolites among triatomines.

Venn diagrams represent metabolite frequencies (numbers in circles) and their share among replicates (circles) for each triatomine species (A: T. infestans or Ti, B: R. prolixus or Rp, C: P. megistus or Pm). For example, the sample size of replicate number 1 of T. infestans (Ti1) is n = 1,049; it shares 505 metabolites in common with Ti2 and 481 with Ti3. 258 metabolites are common among the three replicates. (*) is for the average number and standard deviation of sample size among triplicates and (**) is for those parameters concerning common metabolites between replicate pairs.

Figure 3. Representation of relative metabolite distribution.

Most metabolites were found at very low rates (below 0.025%). The distribution extends above 0.05, up to 14%, but was not shown for clarity.

Figure 4. Distributions of metabolite rate differences.

Three examples are given for the distributions of these differences among feces of different triatomine species, i.e., T. infestans vs. R. prolixus (A), T. infestans vs. P. megistus (B) and R. prolixus vs. P. megistus (C). The histograms focus on the significant part of the samples in terms of representativeness, but the values were found in a larger interval. In all panels, ∼95% of pair differences are found between −0.15 and +0.15 (n = 2,086).

Figure 5. Differences in ion frequencies among triatomine replicates.

Plots are given for all replicate combinations considering the following triatomine pairs: T. infestans vs. R. prolixus (A), T. infestans vs. P. megistus (B) and R. prolixus vs. P. megistus (C). Dots between dashed lines are for the metabolites with small differences among pairs of triatomine species. Dots outside the dashed lines are for the metabolites displaying large differences among pairs of triatomine species (at p≤0.05). For plotting convenience, the scale of the y axis has been limited to the interval −1 to +1. Some pairs exist outside this range (data not shown).

Figure 6. Boolean operations on metabolite rate differences.

Venn diagrams are given for all replicate combinations considering the following triatomine comparisons: R. prolixus vs. T. infestans AND P. megistus (A), T. infestans vs. R. prolixus AND P. megistus (B), P. megistus vs. T. infestans AND R. prolixus (C), and all comparisons above (D).

Figure 7. Metabolic classes in the uniform core.

Frequency is given in number of hits per metabolic category in the uniform core. Only metabolic classes with 10 or more hits are displayed.

Figure 8. Metabolic classes in the variable core.

Frequency is given in number of hits per metabolic category in the following comparisons: P. megistus vs. T. infestans and R. prolixus (white bars), R. prolixus vs. T. infestans and P. megistus (gray bars), and T. infestans vs. R. prolixus and P. megistus (black bars). Only metabolic classes with 5 or more hits are displayed.