Effect of IR knockdown and nematode infection on ECD production by ovaries.

<p><i>C</i>. <i>quinquefasciatus</i> females were injected with either control (dseGFP) or CqIR (dsIR) dsRNA and 5 days post-injection were allowed to feed on uninfected or mf infected blood. At 24 and 48h PBM, ovaries were dissected for incubation (6 h) then media was collected for quantification with the ecdysteroid radioimmunoassay. Data from each set with mean and SEM is plotted. Different letters above a given bar indicate a mean that significantly differs (F _{5, 12} = 7.078, P<0.001). One-way ANOVA with multiple comparison and Tukey's multiple comparison tests were used.</p

Effect of IR knockdown and nematode development on mosquito fecundity.

<p><i>C</i>. <i>quinquefasciatus</i> females were injected with either control (dseGFP) or CqIR (dsIR) dsRNA. 5 days post injection eGFP dsRNA injected mosquitoes were fed on uninfected blood (dseGFP-U) and mf-infected blood (dseGFP-I), and dsIR injected females were fed only on infected blood-fed (dsIR-I). Total number of eggs deposited by each set of mosquitoes was counted 7 days PBM. This experiment was replicated twice with different cohorts of mosquitoes and infected blood. Data from each set with mean and SEM is plotted. Different letters above a given bar indicate means that significantly differ (F _{2, 39} = 260.1; P<0.0001). One-way ANOVA and Tukey's multiple comparisons test were used.</p

Additional file 1: Figure S1. of RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells

Summary of the process employed to select I. scapularis genes for RNAi knockdown experiments. Δ ISE6 proteins from the differential proteomic analysis at 36 hpi were analyzed. Proteins were selected based on (1) increased expression level, (2) strength of proteomic support (minimum 2 peptides identified from LC-MS-MS per protein) from proteins identified in Grabowski et al. [4], and (3) orthology to vertebrate/invertebrate proteins; * orthologous proteins identified in published proteomic studies [4–6, 8]. LGTV denotes proteins that exhibited increased expression following LGTV infection and LGTV & UV-LGTV denotes proteins that exhibited increased expression following both LGTV infection and UV-LGTV treatment. + proteins that exhibited increased expression following LGTV infection as compared to UV-LGTV treatment. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576. The prefix “ISCW” denotes VectorBase accession IDs. Figure S2 Effect of pGEM dsRNA concentrations on ISE6 cell viability following transfection for 60 h. X-tremeGENE (Xtr) transfection reagent was used to optimize pGEM dsRNA (RNAi negative control) concentrations in ISE6 cells at 60 h post transfection. Cell viability readings were compared to the Xtr + OptiMEM (Opti) control (gray bar). Red boxes indicate increased or no significant decrease in ISE6 cell viability. RLU560,590, relative light units 560 nm excitation and 590 nm emission. Error bars represent SEM. Statistical analysis was performed using an unpaired t-test between Xtr + Opti control and each pGEM dsRNA concentration. *p value ≤ 0.05 and **p value ≤ 0.01. Results represent 3 technical replicates and 1 biological replicate (multiple biological replicates completed with 10 ng concentration). Figure S3 Effect of transfection with dsRNA on ISE6 cell viability. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576, hypothetical protein; pGEM, pGEM plasmid (negative control; light gray bars); LGTV 3UTR, 3’ UTR of LGTV TP21 strain (positive control; dark gray bars), RLU560,590, relative light units 560 nm excitation and 590 nm emission. ISE6 cell viability following transfection with 10ng dsRNA for 60 h normalized to the negative control pGEM dsRNA. Results represent 2–5 technical replicates and 3 biological replicates. Error bars represent SEM and unpaired t-tests for comparison of cell viability of the negative pGEM control versus each gene of interest. Table S1 T7-tagged primers used to amplify cDNA and synthesize dsRNA. Table S2 Primers used to amplify cDNA for I. scapularis genes of interest by RT-qPCR. Table S3 Enrichment/cluster analysis of ISE6 proteins that exhibited increased expression following LGTV and UV-LGTV treatment. ISE6 proteins with increased expression following LGTV infection and/or UV-LGTV treatment from [4] were searched via DAVID enrichment analysis. For each cluster, the P value represents a modified Fisher Exact P value, and EASE score implemented in DAVID gene enrichment and functional annotation analysis. Enrichment (E) score of ≥ 1.3 is equal to P value of ≤ 0.05. Table S4 Nucleotide similarity of RT-PCR products amplified from I. scapularis and ISE6 cells and IscaW1 gene models. Table S5 Summary of statistically significant values corresponding to figures. (DOCX 329 kb

Effect of insulin receptor RNA interference on <i>W</i>. <i>bancrofti</i> development in <i>C</i>. <i>quinquefasciatus</i>.

<p>A) RT-PCR analysis of <i>insulin receptor</i> expression in tissues of control (dseGFP) and CqIR (dsIR) dsRNA injected <i>C</i>. <i>quinquefasciatus</i> before a blood meal (0), 7, and 10 days post blood meal (PBM). RT-PCR images are representative of three replicates from different biological cohorts. B) Number of microfilariae in hemolymph of control (dseGFP) and CqIR (dsIR) dsRNA injected females 2–4 h PBM. C) Number of infective L3 larvae in thorax and head of control (dseGFP) and CqIR (dsIR) dsRNA injected females 13 days PBM.</p

Knockdown of MIR expression in the midgut of females delays late trypsin expression and activity after blood ingestion.

<p>(A) Left panel: rqRT-PCR analysis of <i>MIR</i> transcript abundance in mosquito midguts 0–48 h pbm from females (blood-fed 6 days old) treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA at 1 day post eclosion. Transcript levels were standardized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020401#pone-0020401-g001" target="_blank">Fig. 1C</a> with each treatment replicated four times using samples of midguts collected from four females. Right panel: The immunoblot shows MIR expression in midguts (4 midguts per lane) collected 0–48 h pbm from females treated as above after SDS-PAGE, transfer to nitrocellulose, and detection with an MIR antibody. (B) rqRT-PCR analysis of <i>AaET</i> transcript abundance in midguts 2 h pbm from females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. Transcript levels were standardized to 1 for <i>dsEGFP</i>-treated females (negative control) while transcript abundance for <i>dsMIR</i>-treated females is expressed relative to the negative control. Each treatment was replicated three times using samples of midguts collected from four females. (C) rqRT-PCR analysis of <i>AaLT</i> abundance in midguts 0–72 h pbm from females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. Transcript levels were standardized to 1 for the 0 h pbm samples from <i>dsEGFP</i>-treated females (negative control) while transcript abundance for the other samples is expressed relative to the negative control. (D) rqRT-PCR analysis of <i>AaSPVI</i> abundance in midguts 0–72 h pbm from females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. Transcript levels were standardized as in C. (E) Trypsin activity in midguts 24–72 h pbm in females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. A minimum of 5 midguts was individually assayed per treatment. Different letters above a given treatment indicates means that significantly differ (F_{5, 75} = 20.2, P<0.001) followed by comparison of all pairs of means using the Tukey-Kramer procedure, α = 0.05).</p

Amino acids enhance ILP3 stimulation of ovarian ECD production in vitro.

<p>The left side of the graph shows the amount of ECDs produced by ovaries (4 ovary pairs per sample) from non-blood fed females after a 6 h incubation in saline (Sal), amino acid supplemented saline (AA), saline plus 20 pmol ILP3 (sal + ILP3), saline plus ILP3 and 100 pmol rapamycin (sal+ILP3+Rap), amino acid saline plus ILP3 (AA+ILP3) or amino acid saline plus ILP3 and rapamycin (AA+ILP3+Rap). The right side of the graph shows the amount of ECDs produced by ovaries from females injected with <i>dsMIR</i> or <i>dsEGFP</i> RNA (day 1) and blood fed on day 6. Ovaries were then dissected 24, 48 and 72 h pbm and incubated for 6 h in saline. The immunoblot above the graph shows ovary extracts (2 ovaries per lane) probed with an anti-MIR antibody. Each lane on the blot corresponds to the treatment shown directly below on the graph. ECD amounts were determined by RIA using a minimum of four independent samples for each treatment. Overall, ECD amounts vary among treatments (F_{11, 98} = 81.8, P<0.0001). Different letters above a given bar indicate means that significantly differ (Tukey-Kramer procedure, α = 0.05).</p

Amino acids enhance ILP3 stimulation of <i>AaSPVI</i> expression in midguts in vitro.

<p>Midguts were dissected from non-blood fed females and incubated for 3 h in saline (Sal), amino acid supplemented saline (AA), saline plus 20 pmol ILP3 (sal + ILP3), saline plus ILP3 and 100 pmol rapamycin (sal+ILP3+Rap), amino acid saline plus ILP3 (AA+ILP3) or amino acid saline plus ILP3 and rapamycin (AA+ILP3+Rap). rqRT-PCR analysis was then performed to determine relative transcript abundance of <i>AaSPVI</i>. Transcript levels were standardized to 1 for the saline-treated sample (negative control) while transcript abundance for the other treatments is expressed relative to the negative control. Each treatment was replicated three times using midguts from four females.</p

ILP3 rescues midgut trypsin activity and late trypsin expression in blood-fed, decapitated females.

<p>(A) Light micrograph of midguts dissected from an intact and decapitated female at 16 h post blood meal (pbm). The dark red color of the blood bolus from the intact female indicates normal digestion, whereas the bright red color of the blood bolus from a decapitated female indicates delayed digestion. (B) Trypsin-like activity at 24 h pbm in the midgut of blood-fed intact females (positive control) compared to blood-fed, decapitated females injected with saline (Decap), 20 pmol of ILP3 (Decap + ILP3), or 0.1 µg–1 µg of 20-hydroxyecdysone (20E) (Decap + 20E). Different letters above a given treatment indicate means that significantly differ from the intact positive control (F_{5, 83} = 14.0, P<0.001; followed by comparison of means to the control using Dunnett's multiple comparison procedure, α = 0.05). (C) rqRT-PCR analysis of <i>AaLT</i> and <i>AaSPVI</i> expression in midguts at 24 h pbm from intact females and decapitated females injected with saline, 20 pmol ILP3, or 1 µg 20E. Transcript levels are standardized to a level of 1 for midguts from intact females, while transcript levels for other treatments are expressed relative to the intact midgut control. Each treatment was replicated four times using samples of midguts collected from four females.</p

Knock down of <i>MIR</i> expression delays vitellogenin (Vg) expression and reduces egg maturation in blood-fed females.

<p><b>(</b>A<b>)</b> Immunoblot of fat body (0.05 abdomen pelt/lane) collected 0–72 h pbm from females injected with <i>dsEGFP</i> or <i>dsMIR</i> RNA, extracted as 2 pelts, and subjected to SDS-PAGE followed by transfer to nitrocellulose and detection with an Vg antibody. <b>(</b>B<b>)</b> Yolk deposition per oocyte 24–72 h pbm in females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. Different letters above a given bar in the graph indicate means that significantly differ (F_{5, 211} = 186.7, P<0.0001, followed by comparison of all pairs of means using the Tukey-Kramer procedure, α = 0.05). (C) Number of ovary follicles with ≥60 µm of yolk 24–72 h pbm in females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA. Different letters above a given bar in the graph indicate means that significantly differ (F_{5, 195} = 60.1, P<0.0001, followed by comparison of all pairs of means using the Tukey-Kramer procedure, α = 0.05). <b>(</b>D<b>)</b> Number of eggs laid by females treated with <i>dsEGFP</i> or <i>dsMIR</i> RNA (F_{1, 61} = 140.0, P<0.0001).</p

<i>dsMIR</i> RNA plus rapamycin treatment strongly reduces midgut trypsin activity and yolk deposition in blood-fed females.

<p>(A) Trypsin activity 24–72 h pbm in the midgut of females treated with <i>dsEGFP</i> RNA, <i>dsTOR</i> RNA, <i>dsEGFP</i> RNA plus rapamycin, or <i>dsMIR</i> RNA plus rapamycin. Different letters above a given treatment indicate means that significantly differ (F_{10, 123} = 35.5, P<0.0001) followed by comparison of all pairs of means using the Tukey-Kramer procedure, α = 0.05). (B) Yolk deposit per oocyte 24–72 h pbm in females treated with <i>dsEGFP</i> RNA, <i>dsTOR</i> RNA, or <i>dsMIR</i> RNA plus rapamycin. Different letters above a given bar in the graph indicate means that significantly differ (F_{8, 129} = 80.0, P<0.0001 and Tukey-Kramer procedure, α = 0.05).</p