Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/12281
Title: 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
Authors: Dias, Felipe de Almeida
Guerra, Barbara
Vieira, Larissa Rezende
Perdomo, Hugo Diego
Gandara, Ana Caroline Paiva
Amaral, Raquel Juliana Vionette do
Vollú, Renata Estebanez
Gomes, Suzete Araujo Oliveira
Lara, Flavio Alves
Sorgine, Marcos Henrique Ferreira
Medei, Emiliano
Oliveira, Pedro Lagerblad de
Salmon, Didier
Affilliation: Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Centro Nacional de Biologia Estrutural e Bioimagem—CENABIO. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.
Universidade Federal Fluminense. Departamento de Biologia Geral. Niterói, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM). Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Centro Nacional de Biologia Estrutural e Bioimagem—CENABIO. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM). Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.
Abstract: 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. Methodology/Principal Findings 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/Significance 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.
Keywords: Rhodnius prolixus
Trypanosome Life Cycle
Parasite Load
Digestive Tract
DeCS: Reação em Cadeia da Polimerase
Carga Parasitária
Trato Gastrointestinal
Trypanosoma cruzi
Issue Date: 2015
Publisher: Plos One
Citation: DIAS, Felipe de ALmeida; et al. 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. PLoS Negl Trop Dis, v.9, n.10, e0004186, 23p, Oct. 2015.
DOI: 10.1371/journal.pntd.0004186
ISSN: 1935-2727
Copyright: open access
Appears in Collections:IOC - Artigos de Periódicos

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