The ecdysone-induced protein 93 is a key factor regulating gonadotrophic cycles in the adult female mosquito Aedes aegypti

Abstract

Repeated blood feedings are required for adult female mosquitoes to maintain their gonadotrophic cycles, enabling them to be important pathogen carriers of human diseases. Elucidating the molecular mechanism underlying developmental switches between these mosquito gonadotrophic cycles will provide valuable insight into mosquito reproduction and could aid in the identification of targets to disrupt these cycles, thereby reducing disease transmission. We report here that the transcription factor ecdysone-induced protein 93 (E93), previously implicated in insect metamorphic transitions, plays a key role in determining the gonadotrophic cyclicity in adult females of the major arboviral vector Aedes aegypti Expression of the E93 gene in mosquitoes is down-regulated by juvenile hormone (JH) and up-regulated by 20-hydroxyecdysone (20E). We find that E93 controls Hormone Receptor 3 (HR3), the transcription factor linked to the termination of reproductive cycles. Moreover, knockdown of E93 expression via RNAi impaired fat body autophagy, suggesting that E93 governs autophagy-induced termination of vitellogenesis. E93 RNAi silencing prior to the first gonadotrophic cycle affected normal progression of the second cycle. Finally, transcriptomic analysis showed a considerable E93-dependent decline in the expression of genes involved in translation and metabolism at the end of a reproductive cycle. In conclusion, our data demonstrate that E93 acts as a crucial factor in regulating reproductive cycle switches in adult female mosquitoes.

Keywords: autophagy; ecdysone-induced protein 93; juvenile hormone; mosquito; reproduction.

Fig. 1.

The cyclic pattern of E93 expression across the first gonadotrophic cycles. Time course of E93 gene expression during the first cycle including the PE period (6 h and 72 h), FPBM period (6 h, 24 h, 36 h, and 72 h). The expression of E93 at PE 6 h was used as controls during the first cycle. The titers of JH, 20E, and the expression of Vg during the first reproductive cycle are schematically shown at Top. Data are shown as mean ± SEM **P < 0.01.

Fig. 2.

Hairy and Kr-h1 act as intermediate factors in JH/Met repression of the E93 gene. (A) qPCR analysis of E93 transcript abundance after RNAi knockdowns of Kr-h1, hairy, and a mixture of both (Kr-h1 and hairy). (B) The reporter plasmid (E93_1.9kb-Luc) and a control Renilla luciferase reporter vector pCopia were cotransfected into Drosophila S2 cells along with the Hairy-Flag and Gro1-V5 expression plasmids. Western blot results showing the expression of fusion protein Hairy-Flag and Gro1-V5. (C) The Hairy binding site occupancy on the E93 gene promoter region, as measured using ChIP-qPCR. Chromatin was immunoprecipitated with anti-Hairy and anti-IgG (control) antibodies from FBs at 72 h PE. Results are shown as % of total input chromatin compared with IgG control. (D) The E93 coding sequence was used as a control to compare with the enrichment of Hairy binding sites in the E93 promoter region. (E) The Kr-h1 binding site occupancy on the E93 gene promoter region, as measured using ChIP-qPCR. Chromatin was immunoprecipitated with anti-Kr-h1 and anti-IgG (control) antibodies from FBs at 72 h PE. Results are shown as % of total input chromatin compared with IgG control. (F) The E93 coding sequence was used as a control to compare with the enrichment of Kr-h1 binding sites in the E93 promoter region. (G) EMSA results confirming the binding of Kr-h1 to biotin-labeled E93 probe. The unlabeled E93 (cold) probe in 200× molar excess was used to test the binding specificity, while a mutant probe of the same concentration was a control. Histone H3 antibody served as a nonspecific antibody control. Data are shown as mean ± SEM *P < 0.05, **P < 0.01.

Fig. 3.

E93 is the 20E-regulated primary response gene. (A) The occupancy of EcR on the promoter region of the E93 gene as measured using the ChIP-qPCR assay. Chromatin from FBs at 36 h PBM was immunoprecipitated with anti-EcR or anti-IgG (control) antibodies. Results are shown as % of total input chromatin compared with IgG control. (B) The E93 coding sequence was used as a control in the ChIP-qPCR analysis to compare with the enrichment of the EcR-binding region in the E93 promoter. (C) EMSA results showing the binding of EcR to biotin-labeled E93 probe. The unlabeled E93 (cold) probe in 200× molar excess was used to test the binding specificity, while a mutant probe of the same concentration was a control. Histone H3 antibody served as nonspecific antibodies. Data are shown as mean ± SEM **P < 0.01.

Fig. 4.

E93 RNAi negatively affects the development of mosquito ovaries during the first gonadotrophic cycle. Female mosquitoes were injected with dsRNA of E93 or GFP within 24 h PE, and ovarian development was examined at 24 h FPBM. (A) The ovary phenotypes of iGFP and iE93 mosquitoes are shown. Images were captured under a 5-megapixel high-definition CMOS camera built into a Leica EZ4W stereoscopic microscope. (B) The follicle lengths (24 h FPBM) of iGFP and iE93 mosquitoes were measured in ImageJ software. Comparison of the egg deposition (C) and hatchability (D) between iE93 and iGFP mosquitoes. Data shown in B–D are represented as mean ± SEM **P < 0.01.

Fig. 5.

Effect of E93 depletion on the Vg mRNA abundance during the first and second gonadotrophic cycles of female A. aegypti mosquitoes. (A) Female mosquitoes, injected with dsRNA of E93 or GFP within 24 h PE, were given the first blood meal 3 d postinjection. Vg transcript levels were examined at 18 h, 24 h, 36 h, and 44 h FPBM. (B) Another batch of mosquitoes was given a second blood meal after laying eggs, and Vg transcript levels were inspected at 18 h, 24 h, 36 h, and 44 h SPBM. Transcript levels of Vg were quantified using qPCR. Each sample was normalized to its internal control ribosomal protein 7 mRNA (rps7). All the data in iE93 mosquitoes were normalized to that in iGFP mosquitoes, which were represented as 1. (C) Western blotting showing Vg protein levels in iE93 and iGFP mosquitoes at 24 h and 36 h FPBM. Data are shown as mean ± SEM **P < 0.01.

Fig. 6.

Comparison of FB transcriptomes after E93 dsRNA injection between 24 h FPBM and 36 h FPBM. (A) The Venn diagram analysis of commonly and uniquely E93-regulated genes at 24 h and 36 h FPBM. (B) Distribution of gene functional groups within up- and down-regulated E93 RNAi-depleted transcriptome at 24 h and 36 h FPBM. Functional group abbreviations: MOAA, metabolism of other amino acids; MCV, metabolism of cofactors and vitamins; GBM, glycan biosynthesis and metabolism; EIP, environmental information processing; CP, cellular process; XBM, xenobiotics biodegradation and metabolism; Tlat, translation; F/S/D, folding, sorting, and degradation; NM, nucleotide metabolism; AAM, amino acid metabolism; LM, lipid metabolism; and CM, carbohydrate metabolism. Hierarchical clustering analysis of differentially expressed genes involved in translation (C) and vitellogenesis (D) at 24 h and 36 h FPBM between iE93 and iGFP control. Part of the genes associated with ribosome biogenesis and aminoacyl-tRNA biosynthesis are shown in C.

Fig. 7.

Autophagy of mosquitoes is affected by E93. (A) Hierarchical clustering analysis of autophagy-related genes at 36 h FPBM. (B) qPCR analysis of the mRNA levels of autophagy-related genes in E93 RNAi mosquitoes. (C) Immunofluorescence assays showing the ATG8 distribution in FBs of iE93 mosquitoes compared with iGFP control. Anti-ATG8 antibodies were used to detect the ATG8 protein (red). Alexa Fluor 594 was used as the secondary antibody. Nuclei stained with Hoechst 488 was blue. (D) The dual luciferase reporter assay showing the effect of E93 on the ATG8 gene. Cells transfected with the empty vector pAc5.1b or no transfection served as the control. The Renilla luciferase vector pGL4.73 was used as an expression control in the luciferase assays. The Western blot showed the protein levels of E93-V5 fusion proteins after transfection in S2 cells for 48 h using anti-V5 monoclonal antibody. GAPDH antibody was used as a loading control. Data are shown as mean ± SEM **P < 0.01.