Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle

Abstract

BackgroundHere 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.Methodology/Principal FindingsqPCR 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.Conclusions/SignificanceThese 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.

Field-collected Triatoma sordida from central Brazil display high microbiota diversity that varies with regard to developmental stage and intestinal segmentation

“…sordida ’s gut may be explained by the specific function of each segment during blood meal digestion. The anterior midgut of triatomines has a neutral-basic pH near 7.2 and functions as a reservoir of the ingested blood, which remains essentially undigested [ 61 ]. Only water elimination, erythrocyte lysis [ 59 ], and inhibition of blood clotting by anticoagulants [ 62 ] take place.…”

Evaluating the effects of anticoagulants on Rhodnius prolixus artificial blood feeding

“…Adult females were artificially fed on blood collected with heparin at the final concentrations of 2.5–10 U/mL of blood. After 24 h of feeding, the midgut of the insects was dissected and total DNA was extracted following the protocol described by [33]. The DNA sample was subjected to electrophoresis on 0.8% agarose gel.…”

The Influence of Environmental Cues on the Development of Trypanosoma cruzi in Triatominae Vector

“…Midgut colonization by T. cruzi was recently revisited by Ferreira and collaborators, and as reported by Dias (1934), epimastigogenesis was shown to be completed in the PM of R. prolixus (Ferreira et al, 2016) and not in the anterior midgut, as was assumed for quite some time. Furthermore, the AM seems to be an inhospitable environment for the parasite, since the trypomastigote population is severely reduced 24 h after invading this portion of the gut (Dias et al, 2015;Ferreira et al, 2016;reviewed in Guarneri and Lorenzo, 2017). During this initial interval, it is assumed that the remaining parasites differentiate into intermediate or amastigotelike forms and quickly travel to the PM, where they will start to replicate (Guarneri and Lorenzo, 2017).…”