Fig 1.
Protocols used for gene silencing by RNA interference in the fed and starving states.
Protocol 1. Females were injected on day 18 after feeding, fed three days later and dissected on days 5 and 10 (fed state). Protocol 2. Females were injected on day 10 after feeding and dissected 14 days after injection, i.e., on day 24 after the blood meal (starving state).
Fig 2.
Silencing efficiencies of RpAtg8 and RpBmm in the fat body.
Adult females, 18 days after a blood meal, were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (Protocol 1) or injected on the tenth day after feeding and dissected 14 days after injection (Protocol 2) (n = 4-7). (A–B) mRNA levels were determined by qPCR, using Rp18S expression as a reference gene. RpAtg8 and RpBmm mRNA quantification in the fat body. Gene expression levels are relative to each control value (dashed lines). (C) RpAtg8 immunoblotting in the fat body on the 10th day (top image) and the 24th day (bottom image) (n = 3). (D) Total RpAtg8 densitometry on the 10th and 24th days. RpAtg8-I: free RpAtg8. RpAtg8-II: lipidated RpAtg8. All graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, when compared by one-way ANOVA followed by Tukey’s post-test.
Fig 3.
Silenced females accumulate TAG in the fat body during starvation.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (Protocol 1) or injected on the tenth day after feeding and dissected 14 days after injection (Protocol 2). Fat bodies were collected, washed, individually homogenized, and total TAG (A, C, E) and protein content (B, D, F) on the 5th (A, B), 10th (C, D), and 24th (E, F) day after the blood meal were determined. Graphs show mean ± SD (n = 12–23 insects, obtained from 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, when compared by one-way ANOVA followed by Tukey’s post-test.
Fig 4.
Silencing efficiencies of RpAtg8 and RpBmm in the flight muscle.
Adult females, 18 days after a blood meal, were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (Protocol 1) or injected on the tenth day after feeding and dissected 14 days after injection (Protocol 2) (n = 4-7). (A–B) mRNA levels were determined by qPCR, using Rp18S expression as a reference gene. RpAtg8 and RpBmm mRNA quantification in the fat body. Gene expression levels are relative to each control value (dashed lines). (C) RpAtg8 immunoblotting in the flight muscle on the 10th day (top image) and the 24th day (bottom image) (n = 3). (D) Total RpAtg8 densitometry on the 10th and 24th days. RpAtg8-I: free RpAtg8. RpAtg8-II: lipidated RpAtg8. All graphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, when compared by one-way ANOVA followed by Tukey’s post-test.
Fig 5.
Silenced females accumulate TAG in the flight muscle during starvation.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (Protocol 1) or injected on the tenth day after feeding and dissected 14 days after injection (Protocol 2). Flight muscles were collected, washed, individually homogenized, and total TAG (A, C, E) and protein content (B, D, F) on the 5th (A, B), 10th (C, D), and 24th (E, F) day after the blood meal were determined. Graphs show mean ± SD (n = 12–23 insects, obtained from 3 independent experiments). *p < 0.05, **p < 0.01, ****p < 0.0001, when compared by one-way ANOVA followed by Tukey’s post-test.
Fig 6.
Silencing of RpAtg8 and RpBmm affects the expression of lipid metabolism-related genes in the fat body.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (first protocol) or injected on the tenth day after feeding and dissected 14 days after injection (second protocol). Gene expression levels in the fat body were determined by qPCR using specific primers designed to target different genes. Rp18S amplification was used as an endogenous control. Gene expression levels are relative to each control value (dashed line). The graphs show mean ± SEM of 5 independent determinations, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, when compared by Student’s t-test. Akhr, adipokinetic hormone receptor; ACC, acetyl-CoA carboxylase; DGAT1, diacylglycerol acyltransferase 1; DGAT2, diacylglycerol acyltransferase 2; Plin, perilipin.
Fig 7.
Silencing of RpAtg8 and RpBmm affects the expression of lipid metabolism-related genes in the flight muscle.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (first protocol) or injected on the tenth day after feeding and dissected 14 days after injection (second protocol). Gene expression levels in the flight muscle were determined by qPCR using specific primers designed to target different genes. Rp18S amplification was used as an endogenous control. Gene expression levels are relative to each control value (dashed line). The graphs show mean ± SEM of 5 independent determinations, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, when compared by Student’s t-test. Akhr, adipokinetic hormone receptor; ACC, acetyl-CoA carboxylase; DGAT1, diacylglycerol acyltransferase 1; DGAT2, diacylglycerol acyltransferase 2; Plin, perilipin.
Fig 8.
Silencing of RpAtg8 and RpBmm affects physiological aspects of silenced insects.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected on different days after feeding. (A) On different days after the blood meal, insects were dissected and total protein content in the midgut was determined. Symbols represent mean ± SD (n = 10–13). (B) Total protein concentration in the hemolymph was measured on day 5 after feeding. Results are shown as mean ± SD (n = 5–7), compared by one-way ANOVA followed by Tukey’s post-test (C–D) Protein profile of the hemolymph (0.7 μL) by SDS-PAGE (7.5% polyacrylamide) followed by Vg protein densitometry on day 5 after feeding. An egg sample was used to confirm the identification of Vg bands. Arrowheads indicate Vg subunits. Results are shown as mean ± SD (n = 8), compared by one-way ANOVA followed by Tukey’s post-test. (E) Oviposition rates monitored daily after the blood meal. Results show mean ± SD (n = 17). (****): p < 0.0001 analyzed by two-way ANOVA followed by Sidak’s post-test. (F) Total hatching rates were determined, n = 855. (****): p < 0.0001 analyzed by the χ2 test. (G) Survival was monitored daily (n = 18-19, in three independent experiments), being (*) and (****): p < 0.05 and p < 0.0001, respectively, after analysis by the Log-rank test (Mantel-Cox).
Fig 9.
Dual silencing of RpAtg8 and RpBmm leads to additive alterations in LDs diameters in the fat body.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected either five or ten days after feeding (Protocol 1) or injected on the tenth day after feeding and dissected 14 days after injection (Protocol 2). Lipid droplets (LDs) from freshly dissected fat bodies on days 5, 10, and 24 after the blood meal were stained with Nile Red and observed under a confocal laser scanning microscope. DAPI-stained nuclei were also observed. Images are representative from three independent experiments (n = 3). Bars: 40 µm.
Fig 10.
Quantifications of the additive alterations in LDs diameters in the fat body.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later, and dissected five or ten days after feeding (Protocol 1), or injected on day ten after feeding and dissected 14 days after injection (Protocol 2). (A) Histogram of LD diameter distribution and quantification of maximum LD diameters on day 5. (B) Histogram of LD diameter distribution and quantification of maximum LD diameters on day 10. (C) Histogram of LD diameter distribution and quantification of maximum LD diameters on day 24. Three experiments were performed using insects from different cohorts (n = 3), and 2 images from each experiment were quantified. The graphs show the medians ± 5th-95th percentiles of at least 2000 LDs per condition. ****p < 0.0001, when compared by one-way ANOVA followed by Tukey’s post-test.
Fig 11.
Dual silencing of RpAtg8 and RpBmm leads to reduced flight capacity.
Adult females (18 days after a blood meal) were injected with 1 μg of dsRNA for RpAtg8, RpBmm, RpAtg8 + RpBmm, or Mal (control), fed three days later (Protocol 1), or injected on day ten after feeding (Protocol 2). (A) Following protocol 1, insects were subjected to a forced flight test on day 10 after feeding. The duration of flight activity until exhaustion was recorded individually (n = 7-9). (B) Following protocol 2, insects were subjected to a forced flight test on day 24 after feeding. The duration of flight activity until exhaustion was recorded individually (n = 8-9). Graphs show mean ± SDs. **p < 0.01 and ****p < 0.0001 when compared by one-way ANOVA followed by Tukey’s post hoc test.