Figure 1.
HR3-knockdown Aedes mosquitoes showed a delay in impaired vitellogenin (Vg) mRNA and Vg protein levels at the end of vitellogenesis in the fat body.
(A–C) Female mosquitoes were injected with 1 µg dsHR3 or dsMal RNAi, as described in Materials and Methods. HR3 (A and B) and Vg (C) mRNA expression was measured using qPCR at the indicated time points after blood feeding (PBM). Point 0h represents 72h post-eclosion in female mosquitoes prior to blood feeding; this was used as a reference control. Data are expressed as fold induction relative to S7. Data (means + standard errors of the means) from three independent experiments are shown. *Indicates statistical significance <0.05. (B) Magnification scale of the 36-h and 42-h PBM time points from the same experiment shown in A to demonstrate details of the HR3 depletion at the end of the vitellogenic cycle. (C) Monitoring the Vg transcript abundance in the same experiments as in A. A high level of the Vg transcript was observed in FBs of HR3-depleted female mosquitoes at 36 h PBM showing a delayed high expression of the Vg gene. (D) Western blot analysis of Vg protein during several time points of the first gonadotrophic cycle in the fat body of female mosquitoes treated with dsMal or dsHR3 RNAi. A total of 0.01 fat body equivalents was loaded in each lane for SDS/PAGE. A mixture of nine monoclonal antibodies against Vg small subunit was used for Vg protein detection (upper panels). Lower panels with Comassie-stained SDS/PAGE gels used for the western blot experiment are presented as loading controls. Three independent replicates of the experiment were made.
Figure 2.
HR3 inhibits 20E-dependent activation of the A. aegypti Vg gene promoter in luciferase reporter assays.
(A) Drosophila KC-L57-3-11 cells were transiently transfected with 300 ng of the reporter construct pGL3.Vg2100 (carrying 2.1 Kb of the A. aegypti Vg promoter) and the expression plasmids pAc5.EcR (0.2 microg) and pAc5.USP (0.2 microg), indicated as EcR/USP. The expression plasmid pAc5.HR3 (HR3) was added at 0.2, 1 or 2 microg. Expression assays were conducted in the presence (+) or absence (−) of 1 µM 20E for 36 h. 50 ng of the control expression vector pRLCMV. Renilla luciferase was co-transfected for normalization. (B) The assay was conducted in a similar manner, but with the addition of 0.2 µg of the expression plasmid pAc5.E74B (E74B). The expression plasmid pAc5.HR3 was added at 1 or 2 microg. Relative luciferase activity represents ratios of firefly luciferase to Renilla luciferase readings. Values are the mean of three replicates (± SEM). The experiment was repeated three times. *Indicates statistical significance <0.05.
Figure 3.
Effect of HR3 RNAi depletion on mRNA abundance levels of genes involved in 20E response in the fat body of Aedes female mosquitoes.
Female mosquitoes were injected with 1 µg of dsHR3 or dsMal. Transcript levels of Vg, E74B, EcR-A, EcR-B, USP-A, USP-B, betaFTZF1-A, and betaFTZF1-B were quantified by means of qPCR during a time-course experiment that covered from previtellogenesis (PV) to 36 h PBM. Each time point represents the average (± SEM) of three groups of three FBs. Each sample was normalized to its internal control ribosomal protein-7 mRNA. Three independent replicates of the experiment were assayed with three different cohorts of mosquitoes. *Indicates statistical significance <0.05.
Figure 4.
HR3 RNAi depletion impaired the programmed fat body autophagy at the end of the vitellogenic cycle.
(A) Lysotracker and DAPI staining of adult female FBs, 36 h PBM, treated with 1 µg of dsMal or dsHR3 RNAi. Newly emerged female mosquitoes were injected, and five days later they received a blood meal. FBs were dissected 36 h PBM and incubated for 5 min in a solution containing 200 nM of LysoTracker Red DND-99 and 0.01 microg/microl of DAPI. The scale bar is 50 µm. (B) mRNA levels of two genes involved in fat body autophagy at the end of the first vitellogenic cycle in the fat body of Aedes female mosquitoes. qPCR was performed as described before using specific primers for Atg8 and Debcl genes. Each sample was normalized to its internal control ribosomal protein-7 mRNA and three independent experiments were assayed. *Indicates statistical significance <0.05.
Figure 5.
A delay in a timely shutdown of the TOR signaling in fat bodies of HR3-depleted Aedes female mosquitoes.
(A) Western blot analysis of phospho-S6-Kinase (S6K-P) and S6-Kinase (S6K) proteins during the termination of vitellogenesis in the fat body of Aedes female mosquitoes. One fat body equivalent per lane was loaded in a SDS/PAGE gel. The experiment was done in triplicate. (B) Densitometry measurements of three independent experiments from A showing higher levels of S6K-P in dsHR3 mosquitoes. Relative units normalized to its corresponding S6K levels. *Indicates statistical significance <0.05. (C) mRNA levels of LK6-Kinase (LK6) during the first vitellogenic cycle in FBs of female mosquitoes treated with dsMal or dsHR3 RNAi. qPCR was done as previously described. *Indicates statistical significance <0.05.
Figure 6.
The effect of the HR3 RNAi depletion on transcript abundance of HR3 and Vg in the fat body of Aedes female mosquitoes during the first and second gonadotrophic cycles.
Five days after RNAi injection (dsMal as a control or dsHR3), mosquitoes were given a blood meal. Mosquitoes were dissected at the indicated time points as described in Materials and Methods. To study the second vitellogenic cycle, mosquitoes received a second BM 120 h after the first. Each time point represents the average (± SEM) of three groups of three FBs. Each sample was normalized to its internal control ribosomal protein-7 mRNA. Values represent an average of three groups of three FBs. Each experiment was repeated three times (± SEM). *Indicates statistical significance <0.05. Transcript abundance of HR3 (A) and Vg (B) in FBs in vivo. (C) Fat bodies from Aedes female mosquitoes injected with either dsMal or dsHR3 were dissected 96 h after the first blood feeding. They were incubated for 6 h in the presence (20E) or absence (CM) of 1 microM 20E. Vg relative mRNA levels were measured by means of qPCR. Each time point is the average (± SEM) of three groups of three FBs. Samples were normalized to their internal control ribosomal protein-7 mRNA. The experiment was repeated three times with different cohorts of mosquitoes. *Indicates statistical significance <0.05.
Figure 7.
The effect of HR3 RNAi depletion on transcript levels of genes involved in the 20E response in the fat body of Aedes female mosquitoes during the first and the second gonadotrophic cycles.
Transcript abundance of EcRA, EcRB, USPA, USPB, betaFTZ-F1A, and betaFTZ-F1B is represented. Five days after RNAi injection (dsMal as control or dsHR3), mosquitoes were given a blood meal. Mosquitoes were dissected at the indicated time points, as described in Materials and Methods. To study the second vitellogenic cycle, mosquitoes received a second blood meal 120 h after the first blood meal and egg laying. Each time point represents the average (± SEM) of three groups of three FBs. Each sample was normalized to its internal control ribosomal protein-7 mRNA. Values represent an average of three groups of three FBs. Each experiment was repeated three times (± SEM). *Indicates statistical significance <0.05.
Figure 8.
Effect of HR3 RNAi depletion on ovarian development in the second gonadotrophic cycle in Aedes female mosquitoes.
A. Representatives of ovaries from iHR3, iMal and untreated wild type female mosquitoes at 24 h PBM. Note that in iHR3 female mosquitoes ovaries are mostly underdeveloped, or lack visible yolk mass (white). Ovaries from iMal mosquitoes were normally developed similar to those of the wild type untreated control mosquitoes. C. Number of eggs per female mosquito in the second gonadotrophic cycle. iMal - HR3 RNA depleted, iMal – dsRNA Mal-treated, and wt - wild type female mosquitoes. The experiment was repeated three times with different cohorts of mosquitoes. *Indicates statistical significance <0.05.
Figure 9.
A schematic representation of the 20E regulated events during the first vitellogenic cycle in the fat body of the mosquito A. aegypti.
After a blood meal activation, a high level of 20E acts via EcRA/USPB heterodimer activating early genes, BrZ2, E74B and E75A, which synergistically activate late genes such as Vg [15]–[18]. TOR, activated by amino acids and insulin, is essential for activating late genes [3], [6]. TOR is also involved in inhibition of the programmed autophagy during vitellogenesis [19]. HR3 is inhibited by E75A [18], but activated by EcRA/USPB ensuring its timely expression. At the termination time, lowering of the 20E titer results in repressive action of BriZ1 (not shown) and BrZ4 on late genes such as Vg [15], [17]. In this study, we have shown that HR3 is involved in inhibition of late target genes expression (Vg). On the other hand, HR3 activates the EcRB/USPA heterodimer. EcR has been shown to repress TOR and activate autophagy [19]. We postulate that it is the EcRB/USPA heterodimer that is responsible for these functions mediating action of HR3; however, this link requires additional confirmation. HR3 acts as an activator of betaFTZ-F1 that in turn is essential for maintaining cyclicity of egg development. The green ovals – EcR/USP 20E heterodimeric receptor; blue boxes – genes that encode 20E regulated transcription factors (early genes); purple boxes – Target-of Rapamycin; black boxes – autophagy; orange boxes – late target genes. Green arrows depict activating effects and red lines - repressive effects. The basics of the scheme were adapted from [46]for comparison reasons.