Locomotor activity of control (blue line) and infected (red line) <i>Ae. aegypti</i> females under LD 12∶12.

<p>Lines represent the hourly mean activity (+/− SEM) of control and infected females in the four experiments, normalized to the peak of activity of each respective control. The grey shadow represents the dark phase. ZT is the Zeitgeber Time. Light turns on at ZT 0 and turns off at ZT 12. Panel (A) shows a full LD cycle while panel (B) shows only the photophase.</p

Activity increase in Dengue infected <i>Aedes aegypti</i> females.

<p>*The numbers in parenthesis refer to the activity excluding the light-on/light-off transition.</p

Immunolocalization of ABC transporters in the tick midgut digest cell.

<p>Fully engorged females were dissected, fixed and included in paraffin. Deparaffinized sections were stained using polyclonal antibodies against an aminoterminal segment of human PgP-1 (A-C) or rabbit non-immunized serum (D-F), followed by Alexa 633-labeled secondary antibodies. Images are DIC (A and D), fluorescence (B and E) and DIC merged with fluorescence (C and F). Asterisk shows a labeled digestive vesicle. Arrow indicates a labeled digestive vesicle membrane. The scale bars is 40 μm.</p

ABC transporter silencing impairs Zn-Pp IX traffic in digest cells.

<p>Partially engorged females were collected from cattle and were artificially fed with blood supplemented with dsABC (A-C), with Zn-Pp IX plus dsCont (D-F) or with Zn-Pp IX plus dsABC (G-I). In all cases, the blood meal contained 0.5% DMSO (v/v). After 72 h ABM, digest cells were detached from the tissue, and differential interference contrast (DIC) (A, D and G) and Zn-Pp IX fluorescence images (C, F and I) were acquired. Merged images are shown in B, E and H. The white arrows indicate hemosomes (small vesicles) exhibiting a Zn-Pp IX signal in panels A_F; in panels G-I, some hemosomes are indicated, but with no fluorescence associated; white asterisks show digestive vesicles within the Zn-Pp IX signal. The scale bars are 20 μm in all images. The ratio of the number of Zn-PP positive hemosomes to digestive vesicles was measured in 15 randomly chosen images from each condition, obtained from three independent experiments (J). Data shown are mean ± SEM; * means p value < 0.002 (Student’s <i>t</i> test).</p

Identification of a CsA-sensitive ATPase activity in the hemosome membrane.

<p>(A-B) Digest cells from fully engorged tick females 2 days after blood meal showing hemosome (asterisk) and digestive vesicle (DV). Both hemosomes membranes (black arrows) and digestive vesicles membranes (white arrows) exhibit strong ATPase activity, as revealed by the precipitation of cerium phosphate (that appear as electron-dense precipitates near both membranes); (C-D) section from a distinct midgut diverticulum from the same tick, preincubated with 10 μM CsA for 30 min before the ATPase assay. The scale bar is 200 nm (B) or 100 nm (A, C and D). The images are representative of two independent experiments.</p

Identification and expression of ABC transporters in midgut digest cells.

<p>(A) Phylogenetic analysis based on the alignment of RmABCB10 (bold) with ABC transporters from other organisms. Dm: <i>D</i>. <i>melanogaster</i>, Dp: <i>D</i>. <i>pulex</i>, Hs: <i>H</i>. <i>sapiens</i>, Rm: <i>R</i>. <i>microplus</i>. Accession numbers of the sequences are described in Methods. (B) Proposed scheme showing the secondary structure of the RmABCB10 protein consisting of six transmembrane domains (Tm1 to Tm6) and one cytosolic nucleotide-binding domain (NBD). (C) Predicted tertiary structure based on the amino acid sequence of the RmABCB10 protein based on the known structure of human ATP-binding cassette sub-family B member 10 (2YL4). (D) The relative expression on RmABCB10 was evaluated by qPCR in digest cells from both sensitive and resistant strains dissected 3 days ABM. The mean ± SEM are shown (n = 3); (**) represents p < 0.05 (one-way ANOVA followed by Tukey’s test).</p

CsA-sensitive uptake of Sn-Protoporphyrin IX (Sn-Pp IX) is higher in amitraz-resistant ticks.

<p>Digest cells from tick strains sensitive or resistant to amitraz were incubated in the presence of 100 μM Sn-Pp IX for 2 h. (A,B) Amitraz-resistant strain; (C,D) amitraz-sensitive strain; (E,F) amitraz-resistant strain preincubated with 10 μM CsA; (G,H) amitraz-sensitive strain preincubated with 10 μM CsA. A, C, E and G are DIC images. B, D, F and H are fluorescence images of the metalloporphyrin. Arrows indicate Sn-Pp IX fluorescence associated with hemosomes. The scale bar is 60 μm. (I) Quantitative analysis of Sn-Pp IX uptake measured as fluorescence intensity of digest cells from resistant and sensitive strains (expressed in arbitrary units; AU). Data shown are mean ± SEM from groups of 10 randomly chosen images obtained from three independent experiments; * means p < 0.001 (one-way ANOVA followed by Tukey’s test).</p

Heme traffic pathway in the digest cell of the cattle tick <i>Rhipicephalus microplus</i>.

<p>This proposed model integrates data reported here on the role of ABC transporters and results from previous reports describing the uptake of hemoglobin, followed by export of heme from the digestive vesicle to the cytosol and formation of heme aggregates in the hemosome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134779#pone.0134779.ref006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134779#pone.0134779.ref028" target="_blank">28</a>].</p

Uptake of Rhodamine 123 by midgut digest cells.

<p>Digest cells from fully engorged adult females were obtained as described in the Methods. Digest cells were incubated in the presence of 0.5 μM Rhodamine 123 for 4 h. (A-B) Control; (C-D) cells preincubated with 10 μM CsA. Panels are fluorescence images (A and C) or differential interference contrast (DIC) merged with fluorescence (B and D). Digestive vesicles labeled are indicated in the figure by white arrows. Scale bar is 40 μm. Fluorescence intensity was measured in images from two independent experiments (E). Data shown are mean ± SEM (n = 12). * means p < 0.05 (Student’s t test).</p

Bootstrapped phylogram of <i>Rhodnius prolixus</i> midgut lectins aligned with their best matches to the NR database.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number. The remaining sequences were obtained from GenBank and are annotated with the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank GI number. One thousand replicates were done for the bootstrap test using the neighbor joining test.</p