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. 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.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi.

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 µM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 µM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 µM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 µM) is optimal for bioimaging, whereas a high concentration (200 µM CdTe) could be toxic to cells. Taken together, our data indicate that 2 µM QD can be used for the successful long-term study of the parasite-vector interaction in real time.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 μM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 μM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 μM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 μM) is optimal for bioimaging, whereas a high concentration (200 μM CdTe) could be toxic to cells. Taken together, our data indicate that 2 μM QD can be used for the successful long-term study of the parasite-vector interaction in real time.

Sistema de defesa celular e humoral de Rhodnius prolixus infectados com Trypanosoma rangeli: ativadores do sistema profenoloxidase / Cellular and humural defense system of Rhodnius prolixus infected with Trypanosoma rangeli: activators of the profenoloxidase system

Na presente tese, foram estudados aspectos relacionados ao sistema de defesa celular e humoral de Rhodnius prolixus infectados com epimastigotas curtos e longos de Trypanosoma rangeli. Inicialmente, foram investigados os efeitos da radiação gama (Cs 137) sobre o sistema imune e o desenvolvimento do parasita no inseto vetor... Numa outra etapa do trabalho, foram determinados aspectos da defesa humoral pela produção de espécies de oxigênio reativo, óxido nítrico e sistema profenoloxidase em R. prolixus inoculados com epimastigotas curtos ou longos de T. rangeli cepas H14 e Choachi. Os resultados revelaram diferenças importantes na concentração de nitratos e nitritos, indicando atividade de óxido nítrico, além da geração de outras moléculas citotóxicas, como ânions superóxidos (O2-) e atividade prophenoloxidase (proPO), na hemolinfa de insetos infectados com diferentes formas epimastigotas de T. rangeli... Estes resultados indicam que NADPH-oxidase e óxido nítrico sintase podem estar envolvidas na resposta imune de R. prolixus. Numa terceira etapa, experimentos in vivo e in vitro foram desenvolvidos comparando aspectos da ativação do sistema profenoloxidase na hemolinfa de R. prolixus com a resposta dos insetos à infecção com epimastigotas curtos e longos de T. rangeli. Foi demonstrado atividade fenoloxidásica drasticamente reduzida nos insetos alimentados com sangue contendo epimastigotas curtos ou longos quando desafiados por inoculação com epimastigotas curtos... Finalmente, foram desenvolvidos experimentos para demonstrar que a inoculação de glicoinositolfosfolipídeos (GIPLs), purificados da superfície de membrana de epimastigotas curtos e longos das cepas H14 e Choachi de T. rangeli alteravam a ativação da via proPO na hemolinfa de R. prolixus. Experimentos usando espectrometria de massa (ESI-MS), detectaram variações quantitativas, em relação a carboidratos e mioinositol nos GIPLs, obtidos das diferentes formas parasitárias das duas cepas de T. rangeli. Estas alterações podem ser correlacionadas à ativação diferencial do sistema profenoloxidase de R. prolixus. As implicações de todas estas investigações são discutidas em relação ao desenvolvimento de T. rangeli e sua habilidade em superar o sistema proPO de R. prolixus. Estes resultados não só proporcionam uma melhor compreensão da infecção por T. rangeli no inseto-vetor mas também oferece novas idéias sobre papel do processo imune básico nas interações parasita-vetor.