Substrate delivery method in Rhodnius prolixus infected with Dm28c-luc . On the feeding day, insects ingested an average of 250 μl of blood with trypomastigotes and supplemented with D-luciferin (1 mg/ml), images were obtained after 10 seconds exposure by the CCD camera with binning factor of 2 (A). After feeding, insects (1, 2 and 3) were evaluated after 60 μg of D-luciferin was injected into the hemocoel, directly in the hemolymph (B); and the same insects were evaluated after 150 μg D-luciferin application to the cuticle, onto the insect ventral thorax, followed by 15 minutes incubation to allow the substrate to diffuse throughout the body (C). In both cases, the bioluminescent images were acquired after 5 minutes exposure by the CCD camera and binning factor 4. The bioluminescent spots were selected in the image with the ROI measurement tool and the radiance quantified by Living Image® software (D), (gray square) hemocoel injection (black square) topical application.