RNA as a feasible marker of Trypanosoma cruzi viability during the parasite interaction with the triatomine vector Rhodnius prolixus (Hemiptera, Triatominae)

Abstract

A recurring question concerning Trypanosoma cruzi DNA detection/quantification is related to the fact that DNA amplification, by itself, does not differentiate between viable or dead parasites. On the other hand, RNA can be considered a potential molecular marker of pathogens viability. Herein, we developed a quantitative real-time PCR with reverse Transcription (RT-qPCR) to quantify viable T. cruzi in artificially infected Rhodnius prolixus whilst evaluating differences between DNA and mRNA quantification along the insect midgut during 5, 9, 15 and 29 days after feeding. The RT-qPCR presented an improved performance with linearities ranging from 107 to 102 parasites equivalents and 3 to 0.0032 intestine unit equivalents, and efficiencies of 100.3% and 102.8% for both T. cruzi and triatomine targets, respectively. Comparing both RT-qPCR and qPCR, we confirmed that RNA is faster degraded, no longer being detected at day 1 after parasite lysis, while DNA detection was stable, with no decrease in parasite load over the days, even after parasite lysis. We also observed statistical differences between the quantification of the parasite load by DNA and by RNA on day 15 after feeding of experimentally infected R. prolixus. When assessing different portions of the digestive tract, by RT-qPCR, we could detect a statistically significant reduction in the parasite amount in the anterior midgut. Oppositely, there was a statistically significant increase of the parasite load in the hindgut. In conclusion, for this study parasite's viability in R. prolixus digestive tract were assessed targeting T. cruzi mRNA. In addition, differences between DNA and RNA detection observed herein, raise the possibility that RNA is a potential molecular viability marker, which could contribute to understanding the dynamics of the parasite infection in invertebrate hosts.

Fig 1. Evaluation of TcGAPDH as a housekeeping gene for RT-qPCR assays.

As endogenous housekeeping reference gene, T. cruzi GAPDH gene was evaluated in different T. cruzi strains, belonging to DTU I to VI (A) and in different evolutive forms (B). PCR assays were in triplicate, and data were pooled.

Fig 2. Reportable Range for detection and quantification of viable T. cruzi in Rhodnius prolixus digestive tube by real-time RTqPCR.

The Table 2 indicates the standard curve parameters of the assays performed with distinct set of primers. Ten-fold serial dilutions of cDNA were used to generate the standard curve for each target. (A) Linearity ranging from 10⁶ to 10 parasite equivalents, for the TcGAPDH target (B) Linearity ranging from 3 to 0.0032 triatomine equivalents, targeting the gene correspondent to the 12S region of the ribosomal RNA of triatomines.

Fig 3. Comparison between DNA and RNA detection of viable and non-viable T. cruzi samples.

A) Quantification of T. cruzi DNA by qPCR; B) Quantification of T. cruzi RNA by RT-qPCR; C) T. cruzi quantification by microscopical examination in a growth curve, with parasite counting using a NEUBAUER Chamber. The symbols corresponding to the control (viable) and lysate (non-viable) groups are indicated in each graph. Data represent analysis from an experiment with n = 3. Data were analyzed using student t-test. *p<0,05.

Fig 4. Comparison between three methods for parasite load quantification from insect samples under increasing periods after feeding.

A) Microscopical examination, with parasite counting using a neubauer Chamber; B) Quantification of T. cruzi RNA by RT-qPCR; C) Quantification of T. cruzi DNA by qPCR. Analysis (n = 3) from a pool of 5 digestive tubes of R. prolixus under increasing periods after feeding. Data were analyzed using student t-test. *p<0,05.

Fig 5. Quantification of viable parasite load by RT-qPCR in different portions of the digestive tract from R. prolixus experimentally infected.

Follow-up of parasite development in a pool of 5 anterior midguts (AM), 5 posterior midguts (PM) and 5 hindguts (H) of R. prolixus under increasing periods after feeding. Each time point represents three independent experiments (n = 3). Data were analyzed using student t-test. *p<0,05 and **p<0,01.