Fig 1.
Phylogenetic trees showing the relationship between protein sequences of ecdysone-response genes of R. prolixus and other arthropod species.
Abbreviations: Aa (Aedes aegypti), Dm (Drosophila melanogaster), Td (Thermobia domestica), Gl (Gecarcinus lateralis), Me, (Metapenaeus ensis), Gm (Galleria mellonella), Sl (Spodoptera littoralis), Am (Apis mellifera), Es (Eriocheir sinensis), Tm (Tenebrio molitor), Bm (Bombyx mori), Pc (Phaedon cochleariae), Pc (Psacothea hilaris).
Fig 2.
Ecdysone response genes are expressed in the ovary of R. prolixus adult females.
Tissues from three unfed females of R. prolixus were pooled and separated into the following groups: central nervous system (CNS), anterior midgut (AMG), Malpighian tubules (MT), hindgut (HG), fat body (FB), ovaries (OV), and oviducts (OviD) + spermatheca (Sp). RT-qPCR was used to quantify the transcript expression of E75 (A), E74 (B), BR-C (C), HR3 (D), HR4 (E), and FTZ-F1 (F). The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔCt method using 18S rRNA and β-actin as reference genes. The results are shown as the mean ± SEM (n = 3–5). Statistically significant differences at p < 0.05 are indicated by different letters (One-way ANOVA and Tukey’s post hoc test).
Fig 3.
Temporal transcript expression of ecdysone response genes in ovaries.
Two ovaries were pooled from adult female insects and analyzed from unfed and at 1, 2, 3, 4, 5, and 6 days post blood meal (PBM) and transcript levels were quantified by RT-qPCR. The expression of ecdysone response genes including E75 (A), E74 (B), BR-C (C), HR3 (D), HR4 (E), and FTZ-F1(F) were measured. The y axis represents the fold change in expression relative to unfed (value ~ 1) obtained via geometric averaging using 18S rRNA and β-actin as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔΔCt method. The results are shown as the mean ± SEM (n = 4–6). Statistical analysis was performed with one-way ANOVA and Tukey’s test for post hoc analysis. Means with significant differences (P<0.05) from others are denoted by different letters.
Fig 4.
Effects of knockdown of E75, E74 or FTZ-F1 on transcript expression of Halloween genes (spook, phantom, disembodied, shadow and shade) in the ovary of adult females 4 days post blood meal.
Females were injected with dsRNA and blood fed as described in Materials and Methods. Relative transcript levels were measured using RT-qPCR and analyzed using the 2−ΔΔCt method. Data are means ± SEM (n = 4–7). Rp49 and β-actin were used as reference genes. Statistical analysis was performed by Student’s t‐test. *p < 0.05, **p < 0.01, ns = not significant.
Fig 5.
Effects of knockdown of E75, E74 and FTZ-F1 on expression of the ecdysone receptor in the ovary of adult females 4 days post blood meal.
Adult females were injected with dsRNA and fed on a blood meal as described in Materials and Methods. Relative transcript levels were measured using RT-qPCR and analyzed using the 2−ΔΔCt method. Data are means ± SEM (n = 4–6). Rp49 and β-actin were used as reference genes. Statistical analysis was performed by Student’s t‐test. *p < 0.05, **p < 0.01.
Fig 6.
Effects of knockdown of E75, E74 or FTZ-F1 on transcript expression of ecdysone response genes in the ovary of adult females 4 days post blood meal.
Females were injected with dsRNA and blood fed as described in Materials and Methods. Relative transcript levels were measured using RT-qPCR and analyzed using the 2−ΔΔCt method. Data are means ± SEM (n = 4–7). Rp49 and β-actin were used as reference genes. Statistical analysis was performed by Student’s t‐test. *p < 0.05, **p < 0.01, ****p<0.0001, ns = not significant.
Fig 7.
Effect of knockdown of FTZ-F1 and E75 on the hemolymph ecdysteroid titer in adult females at days 4 and 5 post blood meal.
Bars represent mean ± SEM (n = 3–4). The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔΔCt method. Data are means ± SEM (n = 3–4). Rp49 and β-actin were used as reference genes. Statistical analysis was performed by Student’s t‐test. *p < 0.05.
Fig 8.
Effect of knockdown of E75 mRNA in adult females on egg production.
RT-qPCR was used to quantify the relative transcript levels of Vg1 and Vg2 in the fat body (FB) and the ovary (OV) at 4 d post blood meal (PBM) in dsARG (control) and dsE75-injected females. Vg1 in FB (A), Vg2 in FB (B), Vg1 in ovary (C), and Vg2 in ovary (D). dsE75-injection affects ovarian phenotype in comparison to dsARG-injected controls (E and F) (n = 4). Eggs laid per female over 15 days PBM (G) (n = 15) and egg volume (H) (n = 15). Phenotypes of laid eggs from insects injected with dsARG (I) or injected with dsE75 (J and K) (n = 15). At 4 days PBM, ovaries were dissected, and images were taken. Rp49 and β-actin were used as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔΔCt method. Results are presented as mean ± SEM. Student’s t-test was used for statistical analysis. *p < 0.05; **p < 0.01, ****p < 0. 0001, ns = not significant.
Fig 9.
Effect knockdown of E74 mRNA in adult females on egg production.
RT-qPCR was used to quantify the relative transcript levels of Vg1 and Vg2 in fat body (FB) and ovary (OV) at 4 d post blood meal (PBM) in dsARG (control) and dsE75-injected females. Vg1 in FB (A), Vg2 in FB (B), Vg1 in ovary (C), and Vg2 in ovary (D). dsE74-injection affects ovarian phenotype in comparison to dsARG-injected insects (control) (E and F) (n = 4). Eggs laid per female over 15 days PBM (G) (n = 15) and egg volume (H) (n = 15). Phenotypes of laid eggs from insects injected with dsARG (I) or dsE75 (J-K) (n = 15). At 4 days PBM, ovaries were dissected, and images were taken. Rp49 and β-actin were used as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔΔCt method. Results are presented as mean ± SEM. Student’s t-test was used for statistical analysis. ***p < 0.001, ****p < 0. 0001.
Fig 10.
Effect knockdown of FTZ-F1 mRNA in adult females on egg production.
RT-qPCR was used to quantify the relative transcript levels of Vg1 and Vg2 in fat body and ovary at 4 d post blood meal in dsARG (control) and dsFTZ-F1-injected females. Vg1 in FB (A), Vg2 in FB (B), Vg1 in ovary (C), and Vg2 in ovary (D). dsFTZ-F1-injection affects ovarian phenotype in comparison to dsARG-injected insects (control) (E and F) (n = 4). Eggs laid per female over 15 days PBM (G) (n = 15) and egg volume (H) (n = 15). Phenotypes of laid eggs from insects injected with dsARG (I) and insects injected with dsFTZ-F1 (J-K) (n = 15). At 4 days PBM, ovaries were dissected, and images were taken. Rp49 and β-actin were used as reference genes. The transcript levels were quantified using RT-qPCR and analyzed using the 2−ΔΔCt method. Results are presented as mean ± SEM. Student’s t-test was used for statistical analysis. ***p < 0.001, ****p < 0. 0001.
Fig 11.
The effect of RNAi-mediated knockdown of E75, E74 or FTZ-F1 transcripts on expression of transcripts involved in choriogenesis (Rp30 and Rp45) in the ovary 4 days post blood meal (PBM).
Relative transcript levels of Rp30 and Rp45 were measured using RT-qPCR and analyzed using the 2−ΔΔCt method. The results are shown as mean ± SEM (n = 4–5). Rp49 and β-actin were used as reference genes. Statistical analysis was performed using Student’s t‐test. ****p < 0.0001.