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

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.背景：本研究报道了通过精准测定寄生虫载量的方式，监测克氏锥虫（Trypanosoma cruzi）对长红锥蝽（Rhodnius prolixus）消化道的定植过程，具体采用实时定量PCR（qPCR）结合荧光成像与生物发光成像（BLI）的联用策略。这些互补的检测方法揭示了寄生虫种群定殖昆虫肠道并建立媒介感染所需的关键步骤。 方法学/主要研究结果：对昆虫肠道内寄生虫载量的qPCR分析存在诸多局限性，主要源于肠道消化源性产物的存在——这类产物可降解DNA并进一步抑制聚合酶链式反应（PCR）的进行。为此，我们开发了针对克氏锥虫重复卫星DNA序列的实时PCR策略，以添加至昆虫样本提取物中的外源性异源DNA作为归一化内参，从而精准定量昆虫肠道各段（前中肠AM、后中肠PM及后肠H）的寄生虫载量。结合荧光显微镜、BLI成像及qPCR分析，我们发现：寄生虫在穿行昆虫消化道的过程中，绝大多数会在感染后的前24小时内于前中肠发生裂解，且该过程不依赖于肠道菌群。在这一短暂阶段内，活的寄生虫会穿过后中肠，从而开启感染的建立进程。感染后第3~4天（p.i.），寄生虫种群开始定殖后肠，并于感染后第7天达到载量峰值，随后的两周内该水平得以维持。值得注意的是，再次取食后的两周内，后肠内的寄生虫数量波动相对平稳；而前中肠与后中肠内的寄生虫种群则略有增长，且可能作为增殖型上鞭毛体的储存库。 结论与意义：本研究数据表明，昆虫肠道内存在一套精准调控的种群动态平衡机制，可维持寄生虫非增殖型感染性锥鞭毛体与增殖型上鞭毛体之间的动态平衡，这对于媒介昆虫的传播能力至关重要。