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

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome ( approximately 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods

European clinical guidelines for Tourette syndrome and other tic disorders. Part II: pharmacological treatment

To develop a European guideline on pharmacologic treatment of Tourette syndrome (TS) the available literature was thoroughly screened and extensively discussed by a working group of the European Society for the Study of Tourette syndrome (ESSTS). Although there are many more studies on pharmacotherapy of TS than on behavioral treatment options, only a limited number of studies meets rigorous quality criteria. Therefore, we have devised a two-stage approach. First, we present the highest level of evidence by reporting the findings of existing Cochrane reviews in this field. Subsequently, we provide the first comprehensive overview of all reports on pharmacological treatment options for TS through a MEDLINE, PubMed, and EMBASE search for all studies that document the effect of pharmacological treatment of TS and other tic disorders between 1970 and November 2010. We present a summary of the current consensus on pharmacological treatment options for TS in Europe to guide the clinician in daily practice. This summary is, however, rather a status quo of a clinically helpful but merely low evidence guideline, mainly driven by expert experience and opinion, since rigorous experimental studies are scarce

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

The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges

Abstract Background Hematophagous mosquitoes transmit many pathogens that cause human diseases. Pathogen acquisition and transmission occur when female mosquitoes blood feed to acquire nutrients for reproduction. The midgut epithelium of mosquitoes serves as the point of entry for transmissible viruses and parasites. Results We studied midgut epithelial dynamics in five major mosquito vector species by quantifying PH3-positive cells (indicative of mitotic proliferation), the incorporation of nucleotide analogs (indicative of DNA synthesis accompanying proliferation and/or endoreplication), and the ploidy (by flow cytometry) of cell populations in the posterior midgut epithelium of adult females. Our results show that the epithelial dynamics of post-emergence maturation and of mature sugar-fed guts were similar in members of the Aedes, Culex, and Anopheles genera. In the first three days post-emergence, ~ 20% of cells in the posterior midgut region of interest incorporated nucleotide analogs, concurrent with both proliferative activity and a broad shift toward higher ploidy. In mature mosquitoes maintained on sugar, an average of 3.5% of cells in the posterior midgut region of interest incorporated nucleotide analogs from five to eight days post-emergence, with a consistent presence of mitotic cells indicating constant cell turnover. Oral bacterial infection triggered a sharp increase in mitosis and nucleotide analog incorporation, suggesting that the mosquito midgut undergoes accelerated cellular turnover in response to damage. Finally, blood feeding resulted in an increase in cell proliferation, but the nature and intensity of the response varied by mosquito species and by blood source (human, bovine, avian or artificial). In An. gambiae, enterocytes appeared to reenter the cell cycle to increase ploidy after consuming blood from all sources except avian. Conclusions We saw that epithelial proliferation, differentiation, and endoreplication reshape the blood-fed gut to increase ploidy, possibly to facilitate increased metabolic activity. Our results highlight the plasticity of the midgut epithelium in mosquitoes’ physiological responses to distinct challenges. Graphical Abstrac

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