The challenge of <i>O.</i><i>fasciatus</i> with <i>P. serpens</i> induces the formation of nodules.

<p>(A) Light microscopy of living hemocytes and parasites 6 h post-infection and (B) Giemsa-stained nodules 72 hpi. The scale bars are the same for both figures A and B, they represent 100× magnification; (C) Scanning electron microscopy of nodules 72 hpi. This figure is equivalent to 1,500× magnification.</p

Survival of <i>P.</i><i>serpens</i>-infected <i>O. fasciatus</i>.

<p>Groups of 25 adult insects were either infected with 5×10⁴ parasites or mock-infected with PBS and their survival was monitored twice a day. The mock-infected insects did not die due to the challenge. On the other hand, approximately 13% of the insects infected with parasites died, which was significant (P<0.05). The experiment was conducted twice.</p

<i>P.</i><i>serpens</i> multiplies in <i>O. fasciatus</i> hemolymph.

<p>(A and B) Promastigotes in the process of cell division were observed via Giemsa staining. A. 6 hpi; B. 24 hpi. The arrows indicate parasites undergoing division. (C) The number of flagellates in the hemolymph of <i>O. fasciatus</i> after the inoculation of 5×10⁴ parasites. Each point represents the mean ± standard error of five samples. Each sample was obtained by pooling together the hemolymph of at least three insects.</p

Hemocytes phagocytize <i>P.</i><i>serpens</i>.

<p>(A) The ratio of hemocytes containing phagocytized parasites increased throughout the time course of the infection. Each point represents the mean ± standard error of eight samples; each sample was obtained by pooling together the hemolymph of three insects. The mean values were compared using One-way ANOVA with Dunnett's post test (p<0.05). The mean values of 24, 48 and 72 hpi differed significantly from the mean value of 6 hpi, as indicated by asterisks. (B) The number of parasites within the hemocytes increased during the infection. The hemocytes were fixed followed by Giemsa staining at 6, 24, 48 and 72 hpi.</p

<i>P.</i><i>serpens</i> reaches the salivary gland of <i>O. fasciatus</i>.

<p>(A). Anatomy of the <i>O. fasciatus</i> salivary gland. The arrows show: lateral lobes (L.L.), posterior lobes (P.L.), anterior lobes (A.L.), accessory glands (A.G.), salivary duct (S.D.) and hilus (H). (B). SEM of infected salivary glands at 24 hpi. (C) Salivary glands infected with long slender forms at 72 hpi.</p

The number of circulating hemocytes in <i>O.</i><i>fasciatus</i> hemolymph increases after infection.

<p>The insects (n = 24) were challenge with 5×10⁴ parasites and the cellular density of the hemocytes was determined using a Neubauer hemocytometer chamber. Each bar represents the mean ± standard error of eight samples and each sample was obtained by pooling together the hemolymph of three insects. Alternatively, the insects were mock challenged with PBS. The asterisk indicates that each one of the three bars (infected 24 h, infected 48 h and infected 72 h) has value significantly different from the value obtained for the hemocytes collected from hemolymph of non-infected insects, using One-way ANOVA with Dunnett's post test (p<0.05).</p

Intracellular fate of <i>P.</i><i>serpens</i>.

<p>Infected hemocytes were analyzed at 6 hpi. (A) Parasites were observed in a tight PV, for which the membrane is indicated by the black arrows; (B) parasites were also observed in larger PV (asterisk) filled with the same structures (myelin figures, electron-dense matrix and lipid inclusions) regularly found in the lysosomes (L) of hemocytes; (C) lysosomes previously labeled with the BSA-gold complex (10 nm) were observed around the PV (black arrows). (D) In some sections, it was possible to observe gold particles inside the PV and close to the parasite (black arrow in C). L, lysosomes; P, parasite; F, flagellum. Scale bars: (A, C and D) 1 µm, (B) 200 nm.</p

Multiple <i>P.</i><i>serpens</i> parasites inside the PV.

<p>Infected hemocytes were analyzed at 72 hpi. (A) At least two parasites were observed within a vacuole (asterisk). (B) A parasite dividing as demonstrated by the presence of two basal corpuscles indicated by the arrows. P, parasite; F, flagellum; K, kinetoplast. No parasites being digested were observed at 72 hpi. Scale bars: (A) 1 µm, (B) 500 nm.</p