Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.
qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.
These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.
Although the key aspects of the T. cruzi life cycle were described more than one century ago, the development and interactions of T. cruzi with its vector are poorly characterized. By dissection of different compartments of the triatomine gut (prototype Rhodnius prolixus) (i.e., AM, PM and H) at regular time intervals, we evaluated trypanosome development within the insect using an accurate qPCR assay. qPCR analysis of trypanosomal colonization and clearance dynamics in real-time were confirmed in vivo using both fluorescence and bioluminescence imaging, which revealed massive parasite lysis during the first 24 hours post-feeding (p.f.). After one week, the parasite succeeded in establishing a resident population in each compartment of the gut, albeit at varying levels. From one week after the onset of infection in the AM and PM, some resident forms agglomerated into rosettes, clustering in close association with the vector tissue and constituting potential parasite reservoirs of the bug. For the first time, we have described a methodology to accurately quantify parasites in the insect gut that would be a useful tool for evaluating the impact of RNAi silencing of insect genes during the course of infection by T. cruzi.