Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi

Abstract

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 × 10(7) 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.

Figure 1. Dose- and time-dependent effect of RHBP knockdown, and tissue-dependent effect of CAT knock-down, in adult females.

Females were fed E. coli 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 (T-test, P< 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 (T-test, P< 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 E. coli 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 (T-test, P< 0.05).

Figure 2. Inhibition of molting and reduction in transcription levels of RHBP and CAT in third instar nymphs.

Nymphs were fed blood with E. coli 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 (T-test, P< 0.05).

Figure 3. Physiological effects in adult females fed with E. coli expressing RHBP or CAT dsRNA.

Reduction in oviposition, egg development and circulating apo-RHBP in females fed with E. coli producing RHBP dsRNA. (A) 20 day oviposition cycle. In black the three control groups (blood alone (circle), bacteria without dsRNA (square) and bacteria with ANT dsRNA (triangle)), in white the groups fed bacteria expressing RHBP dsRNA at three different concentrations 3.35 × 10⁷ CFU/mL blood (triangle), 4.02 × 10⁷ CFU/mL blood (rhombus) and 5.4 × 10⁷ CFU/ml blood (circle) (n = 5). Error bars represent SEM of three biological replicates. For one of the replicates, an additional control of uninduced RHBP bacteria was used, the insects (n = 8) showed no visible reduction from the normal number of eggs, ranging between 38–50 eggs/female. (B) Effects of the RHBP dsRNA in the ovaries of adult females of R. prolixus 10 days after feeding: left panel shows a control R. prolixus ovary. Right panel shows the effect of the inhibition of RHBP by feeding blood with bacteria expressing RHBP dsRNA. Bar = 1mm. (C) Reduction of apo-RHBP present in hemolymph seven days after feeding blood alone (ctrl) or 5.4 × 10⁷ CFU/mL blood bacteria expressing RHBP dsRNA (RHBP). Titration with hemin showed a significant reduction of the circulating apo-RHBP in knocked down insects (T-test, P = 0.0088). Bars represent SEM, three biological replicates, 1 representative female/replicate. As additional control of sample integrity, SDS-PAGE was performed to corroborate the protein profile. (D) Oviposition cycles in females fed blood alone (black circle), 5.4 × 10⁷ CFU/mL blood bacteria without dsRNA (black square), bacteria expressing ANT dsRNA (black triangle), and bacteria expressing CAT dsRNA (white triangle), (two biological replicates, n = 6 each). (E) The dehydration phenotype was observed in 20% of the eggs laid by females fed with bacteria expressing CAT dsRNA, at 5.4 × 10⁷ CFU/ml of blood. Bar = 0.2 mm.

Figure 4. Reactive oxygen species and CAT specific activity in midguts of females fed with E. coli HT115(DE3) expressing CAT dsRNA.

Females were fed blood alone, with E. coli 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 E. coli 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 (T-test, P< 0.05).

Figure 5. Reduction in RHBP expression and oviposition in females fed with R. rhodnii producing a RHBP dsRNA hairpin.

(A) Females were fed blood with kanamycin alone (kan) or containing 10⁵ CFU/mL R. rhodnii transformed with pBP2lac without insert (pBP2lac), expressing a RHBP dsRNA hairpin (pBP2lacRHBP) or a random nucleotide hairpin (pBP2lacRN). Error bars represent SEM of three biological replicates (n = 12 each). Asterisk indicates statistically different values (T-test, P< 0.05). (B) Reduction of eggs in one oviposition cycle. Three biological replicates (n = 6 each). Error bars represent SEM. Asterisks denote statistically significant values relative to control fed blood alone (T test, P<0.05).