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.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Felipe de Almeida
Fde A
Dias
Centro Nacional de Biologia Estrutural e Bioimagem-CENABIO, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Barbara
B
Guerra
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Larissa Rezende
LR
Vieira
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Hugo Diego
HD
Perdomo
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Ana Caroline Paiva
AC
Gandara
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Raquel Juliana Vionette do
RJ
Amaral
Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Renata Estebanez
RE
Vollú
Departamento de Biologia Geral, Universidade Federal Fluminense, Rio de Janeiro, Brazil.
Suzete Araujo Oliveira
SA
Gomes
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
Flavio Alves
FA
Lara
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Brazil.
Marcos Henrique Ferreira
MH
Sorgine
Centro Nacional de Biologia Estrutural e Bioimagem-CENABIO, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Emiliano
E
Medei
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Rio de Janeiro, Brazil.
Pedro Lagerblad
PL
de Oliveira
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Didier
D
Salmon
D000818:Animals; D005260:Female; D041981:Gastrointestinal Tract / Q000469:parasitology; D008163:Luminescent Measurements; D061848:Optical Imaging; D059208:Parasite Load; D060888:Real-Time Polymerase Chain Reaction; D012239:Rhodnius / Q000469:parasitology*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*