Influence of heme from meal on the α-glucosidase expression (A) and activity (B) in the <i>R. prolixus</i> midgut.

<p>P – control insects fed on plasma; P + H – insects fed on hemin-enriched plasma (500 μM). Insects were fed on rabbit plasma with and without hemin. Four days after feeding, midguts (n = 20) were dissected in cold 100 mM NaCl. The α-glucosidase activity was determined by measuring the release of ρ-nitrophenolate from ρ-nitrophenyl α-D-glucopyranoside. Results shown are representative of three independent experiments run in triplicate. Plasma plus hemin is significantly different from plasma *(<i>P</i><0.05).</p

Analysis of α-glucosidase genes using Blastx.

<p>Analysis of α-glucosidase genes using Blastx.</p

Physiological effects of dsRNA-mediated silencing of α-glucosidase.

<p>The insects were injected with 2 µL of 100 mM PBS pH 7.4 or dsLacZ (controls) and dsα-glu (2 or 10 µg/female); mortality and oviposition were monitored 4 days after feeding. In all panels, results are means ±SEM (n = 70). The insects injected with 10 µg dsα-glu, analyzed 4 days feeding, were significantly different from control insects injected with both PBS and dsLacZ and also analyzed 4 days after feeding ^*(<i>P</i><0.05).</p>a<p>Access number to GenBank database.</p>b<p>Function expected according the Blastx sequence results similarity in GenBank database.</p>c<p>Protein Data Bank.</p

Hz formation activity in the presence or absence of maltose <i>in vitro</i>.

<p>Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006966#s4" target="_blank">materials and methods</a>. Hz formation activity was expressed as nmol of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(<i>P</i><0.05).</p

Analysis of the α-glucosidase sequence.

<p>A. Alignment of amino acid sequences of α-glucosidase from <i>R. prolixus</i> (AgluRp), <i>Culex quinquefasciatus</i> (AGluCulex, AGluCulex2, AGluCulex3), α-amylase from <i>Aedes aegypti</i> (AmAeds), maltase-like Agm2 from <i>Anopheles gambiae</i> (MaltaseAgm2), maltase 2 from <i>Drosophila virilis</i> (Maltase2Dv), α-Glucosidase from Gsj (AgluGsj), Oligo-1,6-Glucosidase from <i>Bacillus cereus</i> (GluOligo) and α-glucosidase from <i>Saccharomyces cerevisiae</i> (AgluSc). Identical residues are indicated by “*”; conserved and semiconserved residues are indicated by “:” and “.”, respectively. Residues of aspartic acid and histidine present in AGluRp and also present in AGluSc are marked in gray. The secondary structure prediction using the JPred server is represented in red (α-helices), green (β-sheets) and blue (loops). B. Partial nucleotide sequence of the <i>R. prolixus</i> α-glucosidase cDNA and its deduced amino acid sequence. The amino acid sequences used for the design of specific Real Time-PCR primers are underlined.</p

Hz formation and α-glucosidase activities in the presence or absence of inhibitors <i>in vitro</i>.

<p>A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti <i>D. peruvianus</i> α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006966#s4" target="_blank">materials and methods</a>. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol <i>ρ</i>-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(<i>P</i><0.05).</p