Modulation of peritrophin mRNA expression upon <i>Le. major</i> infection.

<p>qRT-PCR assays depicting differences in PpPer1 (<b>A</b>), PpPer2 (<b>B</b>), and PpPer3 (<b>C</b>) mRNA levels between non-infected and <i>Le. major</i> infected midguts dissected at 24 h, 48 h, and 72 h PBM. Each dot (symbol) represents the mRNA expression levels in a single midgut whereas horizontal bars indicate mean expression levels. The cDNA encoding the S3 protein of the 40S ribosomal subunit was used as the housekeeping control gene. The mean expression of non-infected midguts was used as a standard (100%) for comparisons to the percentage of mRNA expression of <i>Le. major</i> infected midguts for each time point. NI: Non-infected. INF: <i>Le. major</i> infected. NS: Not significant. *: Statistically significant, p<0.05.</p

Effect of dsRNA injection on <i>Le. major</i> infection level in <i>P. papatasi</i>.

<p>Parasite load was categorized according to the number of <i>Le. major</i> per midgut. (A) Percentage of sand flies injected with either dsCtr or dsChit1 exhibiting no infection (0 parasites), as well as light (1–1,000 parasites), moderate (1,000–10,000 parasites), or heavy (>10,000 parasites) infection at 48 h PBM. Differences are statistically significant (Chi-square, p = 0.01). (B) Percentage of sand flies injected with either dsCtr or dsChit1 exhibiting either no parasites or light infection (0–1000 parasites), or moderate infection (>1,000 parasites) at 120 h PBM. Differences are statistically significant (Fisher's exact test, p = 0.04). n: Number of flies dissected.</p

dsRNA effect on <i>PpChit1</i> RNA levels.

<p>Real-Time PCR comparing the mRNA level of <i>PpChit1</i> between flies injected with 80.5 ng (A) or 144 ng (B) of dsPpChit1 (dsChit1) or dsControl (dsCtr) double-strand RNAs. Significant <i>PpChit1</i> transcript reduction was exhibited by dsPpChit1 injected flies at 24 h (A and B), 48 h, 72 h, and 96 h PBM (A). <i>PpChit1</i> mRNA levels were normalized with the S3 housekeeping gene. Results are presented as a percent of <i>PpChit1</i> expression levels in dsPpChit1 injected flies over the mean of <i>PpChit1</i> expression levels in dsControl injected flies (considered as 100%) for each time point. The variance in PpChit1 expression in dsControl injected flies is also shown. Each dot represents <i>PpChit1</i> RNA levels in a single fly. Horizontal bars indicate mean expression level. *: Statistically significant at p<0.05.</p

Expression of PpPer1 and PpPer3 in <i>P. papatasi</i> midgut lysates.

<p>Expression of native PpPer1 and PpPer3 in <i>P. papatasi</i> midgut lysates was assessed using one midgut equivalent (from pools of five guts for each time point) of non blood fed and blood fed <i>P. papatasi</i> per lane. Lanes: M, size marker; 1, non blood fed midgut; 2, blood fed midgut dissected 24 h PBM; 3, blood fed midgut dissected 48 h PBM; 4, blood fed midgut dissected 72 h PBM; 5, blood fed midgut dissected 96 h PBM. Proteins were transferred to nitrocellulose and blots were incubated with anti-PpPer1 (A) and with anti-PpPer3 (B) specific antisera. Arrows point the native proteins PpPer1 (A) and PpPer3 (B).</p

<i>PpPer1</i> knockdown at mRNA and protein levels.

<p>(<b>A</b>) real time qRT-PCR showing <i>PpPer1</i> mRNA level reduction in dsRNA-injected (dsPer1) <i>P. papatasi</i> compared to control injected (GFP dsRNA). Knockdown effects in <i>PpPer1</i> mRNA expression were assessed at 24 h and 48 h PBM that corresponded to 72 h and 96 h post dsRNA injection. Each symbol represents mRNA expression levels in a single midgut. Horizontal bars indicate mean expression levels. The S3 gene was used as the housekeeping control gene. The mean expression of <i>PpPer1</i> in dsGFP-injected flies was used as 100% standard. NS: Not significant. *: Statistically significant, p<0.05 (Unpaired t-test). (<b>B</b>) Western blot assay showing reduction in PpPer1 protein levels at 24 h PBM (72 h after dsRNA injection) in dsPer1-injected flies compared to dsGFP-injected (chemiluminescence development). Nine and a half micrograms of protein were loaded onto 10% NuPAGE gel. (<b>C</b>) Densitometry showing 44% reduction in the intensity of PpPer1 protein band obtained after chemiluminescence development compared to the PpPer1 band intensity of dsGFP-injected flies. For all <i>PpPer1</i> knockdown assays, 80 ng of dsRNA was injected intrathoracically into 3-to-5 day old <i>P. papatasi</i> females fed on 30% sucrose solution <i>ad libitum</i>.</p

Peritrophin mRNA expression profiles.

<p>Expression of <i>PpPer1</i>, <i>PpPer2</i>, and <i>PpPer3</i> mRNAs was assessed by RT-PCR (23–25 cycles) in <i>P. papatasi</i> midguts dissected from adult females at different time points before and after blood feeding (0–144 hours PBM) (<b>A</b>); in pools of tissues other than midgut (<b>B</b>); and in eggs (pool of 20 eggs) or whole body of larvae and pupae (<b>C</b>). β-Tub was used as the housekeeping control gene. The size of the cDNA fragments amplified were 122 bp (<i>PpPer1</i>), 168 bp (<i>PpPer2</i>), 121 bp (<i>PpPer3</i>), and 148 bp (β-Tub). CC: Carcass. HG: Hindgut. FB: Fat Body. HS: Head along with salivary glands. OV: Ovaries. MT: Malpighian Tubules. E: Eggs. L1: Larval stage 1. L2: Larval stage 2. L3: Larval stage 3. L4: Larval stage 4. P: Pupa. (-): Negative control.</p

dsRNA effect on <i>Le. major</i> development.

<p>Intra-thoracic injections of dsPpChit1 (80.5 ng) reduce <i>Le. major</i> load in <i>P. papatasi</i> midgut. (A). At 48 h PBM, <i>Le. major</i> density was reduced on average 46% in dsPpChit1 (dsChit1) injected compared with dsControl (dsCtr) injected. (B). <i>Le. major</i> parasites per midgut were further reduced at 120 h PBM in dsPpChit1 injected flies, reaching on average 63% reduction over the dsControl injected ones. Each dot represents parasite number in a single <i>P. papatasi</i> midgut. Horizontal bars display mean parasite numbers. n: Number of flies analyzed. *: Statistically significant at p<0.05. Graphs represent one similar result of two independent experiments.</p

Effects of PpPer1 knockdown on <i>Le. major</i> infection.

<p>Knockdown of PpPer1 leads to greater <i>Le. major</i> load in the midgut of <i>P. papatasi</i>. At 48 h post-infection (<b>A</b>), dsPpPer1 (dsPer1) injection caused an increased (39%; p<0.05 Mann-Whitney U test) in <i>Le. major</i> load compared to dsGFP-injected <i>P. papatasi</i>. Results shown are from combined data of two independent experiments. (<b>B</b>) Although not statistically significant, <i>PpPer1</i> knockdown led to 22% increase in <i>Le. major</i> load in <i>P. papatasi</i> midguts at 96 h post-infection when compared to dsGFP-injected. Each dot (filled circle or square) represents number of <i>Le. major</i> in a single midgut whereas horizontal bars indicate mean parasite number. <i>P. papatasi</i> were infected with 5×10⁶<i>Le. major</i> amastigotes/ml in heat-inactivated mouse blood. N: Number of <i>P. papatasi</i> midguts dissected. NS: Not significant. *: Statistically significant, p<0.05.</p

dsRNA effect on PpChit1 protein levels.

<p>(A). Western blot assay pointing to <i>PpChit1</i> knock down in dsPpChit1 injected flies (80.5 ng dsRNA) at 48 h PBM. (B). Midgut extracts from flies injected with 144 ng dsPpChit1 (dsChit1) displayed weaker bands (56 kDa) than dsControl (dsCtr) injected flies at 48 h and 72 h PBM. A–B, Colorimetric development. (C). Western blot assay depicting strong PpChit1 expression reduction in flies injected with 144 ng dsPpChit1 (dsChit1) compared with dsCtr injected ones at 48 h PBM (Chemiluminescence development). (D). Densitometry analysis of PpChit1 protein bands obtained in the chemiluminescence assay revealing 95% reduction in PpChit1 expression between dsPpChit1 and dsControl injected flies.</p

Chitin binding assay.

<p>Recombinant rPpPer1 and rPpPer2 were assayed for the capacity to bind colloidal chitin. Wash and elute fractions corresponding to PpPer1 (<b>A</b>) and rPpPer2 (<b>B</b>) were loaded onto reducing 4–12% NuPAGE gels. Lanes: M, molecular weight marker; 1, unbound fraction; 2, wash 1 (10 mM sodium phosphate buffer, pH 8.0); 3, wash 2 (10 mM sodium phosphate buffer, pH 8.0 - 1M sodium chloride); 4, wash 3 (0.1 M acetic acid); 5, elute (SDS lysis buffer); 6, purified protein. Proteins were transferred and blots were incubated with anti-His followed by anti-mouse AP-conjugated.</p