title: Bioluminescent imaging of Trypanosoma cruzi infection in Rhodnius prolixus.
abstract: BACKGROUND
  Usually the analysis of the various developmental stages of Trypanosoma cruzi in the experimentally infected vertebrate and invertebrate hosts is based on the morphological observations of tissue fragments from animals and insects. The development of techniques that allow the imaging of animals infected with parasites expressing luciferase open up possibilities to follow the fate of bioluminescent parasites in infected vectors.
  
  
  METHODS
  D-luciferin (60 μg) was injected into the hemocoel of the whole insect before bioluminescence acquisition. In dissected insects, the whole gut was incubated with D-luciferin in PBS (300 μg/ml) for ex vivo bioluminescence acquisition in the IVIS® Imaging System, Xenogen.
  
  
  RESULTS
  Herein, we describe the results obtained with the luciferase gene integrated into the genome of the Dm28c clone of T. cruzi, and the use of these parasites to follow, in real time, the infection of the insect vector Rhodnius prolixus, by a non- invasive method. The insects were evaluated by in vivo bioluminescent imaging on the feeding day, and on the 7 th, 14 th, 21 st and 28 th days after feeding. To corroborate the bioluminescent imaging made in vivo, and investigate the digestive tract region, the insects were dissected. The bioluminescence emitted was proportional to the number of protozoans in regions of the gut. The same digestive tracts were also macerated to count the parasites in distinct morphological stages with an optical microscope, and for bioluminescence acquisition in a microplate using the IVIS® Imaging System. A positive correlation of parasite numbers and bioluminescence in the microplate was obtained.
  
  
  CONCLUSIONS
  This is the first report of bioluminescent imaging in Rhodnius prolixus infected with trypomastigotes of the Dm28c-luc stable strain, expressing firefly luciferase. In spite of the distribution limitations of the substrate (D-luciferin) in the insect body, longitudinal evaluation of infected insects by bioluminescent imaging is a valuable tool. Bioluminescent imaging of the digestive tract infected with Dm28c-luc is highly sensitive and accurate method to track the fate of the parasite in the vector, in the crop, intestine and rectum. This methodology is useful to gain a better understanding of the parasite - insect vector interactions.
authors.affiliation: Laboratório de Ultraestrutura Celular Hertha Meyer, UFRJ, Rio de Janeiro, RJ, Brazil.
authors.forename: Cristina
authors.initials: C
authors.lastname: Henriques
authors.forename: Daniele P
authors.initials: DP
authors.lastname: Castro
authors.forename: Leonardo H F
authors.initials: LH
authors.lastname: Gomes
authors.forename: Eloi S
authors.initials: ES
authors.lastname: Garcia
authors.forename: Wanderley
authors.initials: W
authors.lastname: de Souza
mesh_terms: D000818:Animals; D054884:Host-Pathogen Interactions; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D012239:Rhodnius / Q000469:parasitology*; D013194:Staining and Labeling / Q000379:methods; D014349:Trypanosoma cruzi / Q000472:pathogenicity*
chemical_list: D008156:Luciferases