title: Detection of viable plasmodium ookinetes in the midguts of anopheles coluzzi using PMA-qrtPCR.
abstract: BACKGROUND
  Mosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density.
  
  
  METHODS
  Quantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form).
  
  
  RESULTS
  By combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut.
  
  
  CONCLUSIONS
  The PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.
authors.affiliation: Department of Life Sciences, Imperial College London, London, UK. t.habtewold@ic.ac.uk.|Department of Comparative Physiology and Biometrics, University of Ghent, Ghent, Belgium. t.habtewold@ic.ac.uk.
authors.forename: Tibebu
authors.initials: T
authors.lastname: Habtewold
authors.affiliation: Department of Life Sciences, Imperial College London, London, UK.|Costello Medical Consulting, Cambridge, UK.
authors.forename: Zoe
authors.initials: Z
authors.lastname: Groom
authors.affiliation: Department of Comparative Physiology and Biometrics, University of Ghent, Ghent, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Duchateau
authors.affiliation: Department of Life Sciences, Imperial College London, London, UK.
authors.forename: George K
authors.initials: GK
authors.lastname: Christophides
mesh_terms: D000818:Animals; D000852:Anopheles / Q000382:microbiology / Q000469:parasitology*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D002470:Cell Survival; D004772:Entomology / Q000379:methods*; D041981:Gastrointestinal Tract / Q000382:microbiology / Q000469:parasitology; D010962:Plasmodium berghei / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*