Slender trypanosomes in a continuously dividing population activate the PAD1 pathway in vivo upon uptake by the tsetse fly. Tsetse flies were infected with either slender or stumpy trypanosomes. Flies were dissected at different timepoints after infection. Living trypanosomes were microscopically analysed in the explanted tsetse midguts and scored for the expression of the fluorescent stumpy reporter GFP:PAD1UTR in the nucleus. Slender cells (n = 1845) are shown in dark grey bars, and stumpy cells (n = 1237) are shown in light grey bars. Slender cell populations continuously divide while turning on the PAD1 pathway, seamlessly transitioning into the first fly form. Total dividing slender population are seen as a green/blue dot. Total PAD1 positive (+) dividing slender populations are shown with a green dotted line. Total PAD1 negative (-) dividing slender populations are shown as a dotted blue line. Stumpy cell populations do not start to divide until 48 after uptake, after they have started to become the first fly form. Total dividing stumpy populations are shown as a dotted gray line.