Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi

Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3) above 3.35 × 107 CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP) and 84% (CAT). Embryogenesis was arrested in 16% (RHBP) and 20% (CAT) of laid eggs. Feeding females 105 CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control

Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP), Heme Oxygenase (HO) and the heme exporter Feline Leukemia Virus C Receptor (FLVCR), components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM), where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of these key proteins after a blood- meal can be extremely deleterious to the insects in their various stages of development

Characterization of horizontally acquired ribotoxin encoding genes and their transcripts in Aedes aegypti

Ribosome Inactivating Proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of the 28S rRNA. The occurrence of RIP genes has been described in a wide range of plant taxa, as well as in several species of bacteria and fungi. A remarkable case is the presence of these genes in metazoans belonging to the Culicinae subfamily. We reported that these genes are derived from a single horizontal gene transfer event, most likely from a bacterial donor species. Moreover, we have shown evidence that mosquito RIP genes are evolving under purifying selection, suggesting that these toxins have acquired a functional role in these organisms. In the present work, we characterized the intra-specific sequence variability of Aedes aegypti RIP genes (RIPAe1, RIPAe2, and RIPAe3) and tested their expression at the mRNA level. Our results show that RIPAe2 and RIPAe3 are transcribed and polyadenylated, and their expression levels are modulated across the developmental stages. Varibility among genes was observed, including the existence of null alleles for RIPAe1 and RIPAe2, with variants showing partial deletions. These results further support the existence of a physiological function for these foreign genes in mosquitoes. The possible nature of this functionality is discussed.Fil: Lapadula, Walter Jesús. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Marcet, Paula L.. Centers for Disease Control and Prevention. National Center for Infectious Diseases. División of Parasitic Diseases; Estados UnidosFil: Taracena, Mabel. Centers For Disease Control And Prevention. National Center For Infectious Diseases; Estados UnidosFil: Lenhart Resourses, Audrey. Centers For Disease Control And Prevention. National Center For Infectious Diseases; Estados UnidosFil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentin

Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult females

Abstract Background Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae. Methods We studied the expression pattern of the dsx gene in the An. gambiae G3 strain. We knocked down AgdsxF expression in larvae through oral delivery of double stranded RNA (dsRNA) produced by bacteria and observed its effects in adults. Results Our results show that feeding of AgdsxF dsRNA can effectively reduce (> 66%) the mRNA of female dsx transcript and that there is a concomitant reduction in the number of female larvae that achieve adulthood. Control groups produced 52% (± 3.9% SE) of adult males and 48% (± 4.0% SE) females, while AgdsxF dsRNA treated groups had 72.1% (± 4.0% SE) males vs 27.8% females (± 3.3% SE). In addition, the female adults produce fewer progeny, 37.1% (± 8.2% SE) less than the controls. The knockdown was sex-specific and had no impact on total numbers of viable male adults, in the male dsx transcripts or male fitness parameters such as longevity or body size. Conclusions These findings indicate that RNAi could be used to improve novel mosquito control strategies that require efficient sex separation and male-only release of An. gambiae by targeting sex determination genes such as AgdsxF. The advantages of using RNAi in a controlled setting for mosquito rearing are numerous, as the dose and time of exposure are controlled, and the possibility of off-target effects and the waste of female production would be significantly reduced

Reactive oxygen species and CAT specific activity in midguts of females fed with <i>E. coli</i> HT115(DE3) expressing CAT dsRNA.

<p>Females were fed blood alone, with <i>E. coli</i> HT115(DE3) expressing ANT dsRNA or CAT dsRNA, at 5.54 × 10⁷ CFU/mL blood. Midguts were dissected six days after feeding, incubated with dihydroethidium (DHE) and photographed under epifluorescence microscopy (Zeiss Observer.Z1 with Zeiss Axio Cam MrM using a filter set 10 (Exc 450–490 nm/ emission 515–565 nm)). Photographs show representative individuals from each group, inserts are differential interference contrast images. (B) Mean specific activity of CAT in insects fed <i>E. coli</i> HT115(DE3) expressing dsRNA CAT. Two biological replicates, n = 3 each. Error bars represent standard error of the mean. Asterisk indicates significant difference from control (<i>T</i>-test, <i>P</i>< 0.05).</p

Dose- and time-dependent effect of RHBP knockdown, and tissue-dependent effect of CAT knock-down, in adult females.

<p>Females were fed <i>E. coli</i> expressing dsRNA. RHBP (A) Expression of RHBP relative to actin on day five after feeding with different amounts of bacteria expressing RHBP dsRNA as compared to insects fed sterile blood: 2.24 × 10⁷ CFU/ml of blood (n = 6), 3.35 × 10⁷ CFU/ml of blood (n = 12) and 5.4 × 10⁷ CFU/ml of blood (n = 8). (B) Expression of RHBP in insects fed 5.4 × 10⁷ CFU/mL blood using bacteria expressing RHBP dsRNA, ANT dsRNA, and without dsRNA. Asterisk indicates statistically different values (<i>T</i>-test, <i>P</i>< 0.05) between experimental groups exposed to bacteria with RHBP dsRNA (n = 6), bacteria with ANT dsRNA (n = 8), bacteria without dsRNA (n = 6), as compared to groups fed blood alone (n = 6). (C) Time-dependent relative expression of RHBP in insects fed 5.4 × 10⁷ CFU bacteria/ml blood ten days after feeding. Asterisk indicates statistically different values (<i>T</i>-test, <i>P</i>< 0.05) between each group and the group fed sterile blood. CAT (D-G) Tissue-specific silencing in females fed with 5.4 × 10⁷ CFU/mL <i>E. coli</i> HT115(DE3) expressing dsRNA CAT or blood alone. (D) Anterior midgut, (E) posterior midgut, (F) fat body and (G) ovaries from each individual were processed to measure expression of CAT, relative to controls. Bars represent SEM, three biological replicates (n = 3 per replicate). In all, asterisk indicates statistically different values as compared to controls fed with blood alone (<i>T</i>-test, <i>P</i>< 0.05).</p

Inhibition of molting and reduction in transcription levels of RHBP and CAT in third instar nymphs.

<p>Nymphs were fed blood with <i>E. coli</i> producing RHBP and CAT dsRNA. (A) Reduction of molting in third instar nymphs fed bacteria producing RHBP or CAT dsRNA as compared with nymphs fed blood without bacteria and with bacteria expressing ANT dsRNA (two biological replicates, n = 10 each). (B) Relative expression of RHBP in third instar nymphs fed bacteria producing RHBP dsRNA (two biological replicates, n = 3 each). (C) Relative expression of CAT in midguts of third instar nymphs fed with bacteria producing RHBP dsRNA (two biological replicates, n = 3 each). Asterisk indicates statistically different values compared with the control fed blood alone (<i>T</i>-test, <i>P</i>< 0.05).</p

General structure of the midgut epithelium of <i>Aedes aegypti</i> and modulation of cell proliferation upon blood meal.

<p>The midgut epithelia from a blood-fed <i>A</i>. <i>aegypti</i> females were fixed in PFA and in <b>(A)</b> sections of 0.14 μm were stained with WGA-FITC (green), red phalloidin (red) and DAPI (blue). The peritrophic matrix (PM), intestinal lumen (Lumen), polyploid enterocytes (EC) and basally localized–putative proliferative cells (*)–are visible. In <b>(B)</b>, confocal image (z-stack of 0.7 μm slides (20X)) of the two monolayers of the midgut of a blood-fed female, 5 days post feeding, stained with PH3 mouse antibody (green), DAPI (blue), and phalloidin (red)–Inset (2x): polyploid enterocytes (EC) are PH3-positive ISC (ISC) are visible. <b>(C)</b> Mosquitoes were fed on a sugar solution (10% sucrose), blood or blood supplemented with 100μM of the pro-oxidant paraquat. The insect midguts were dissected 24 hours after feeding and immunostained for PH3. Representative images of mitotic (PH3-labeled) cells (red) in the epithelial midgut of animals fed on sugar, blood or blood supplemented with paraquat are shown. The nuclei are stained with DAPI (blue). The arrowheads indicate PH3+ cells. <b>(D)</b> Quantification of PH3-positive cells per midgut of sugar, blood or blood plus paraquat-fed mosquitoes for sugar and blood and 18 for blood-paraquat fed midguts. The experiments were performed on Red Eye mosquito strain. The medians of at least three independent experiments are shown (n = 40 for sugar and blood and n = 18 for paraquat supplemented blood). The asterisks indicate significantly different values, **** P<0.0001 (Student’s t-test).</p