Mode of action of methoprene in affecting female reproduction in the African malaria mosquito, Anopheles gambiae

Abstract

Background: One of the most studied actions of juvenile hormone (JH) is its ability to modulate ecdysteroid signaling during insect development and metamorphosis. Previous studies in mosquitoes showed that 20-hydroxyecdysone (20E) regulates vitellogenin synthesis. However, the action of JH and its mimics, e.g. methoprene, on female reproduction of mosquitoes remains unknown.

Results: Here, a major malaria vector, Anopheles gambiae Giles, was used as a model insect to study the action of methoprene on female reproduction. Ecdysteroid titers and expression profiles of ecdysone-regulated genes were determined before and after a blood meal. An ecdysteroid peak was detected at 12 h post blood meal (PBM). The maximum expression of ecdysone-regulated genes, such as ecdysone receptor (EcR), hormone receptor 3 (HR3) and vitellogenin (Vg) gene, coincided with the ecdysteroid peak. Interestingly, topical application of methoprene at 6 h PBM delayed ovarian development and egg maturation by suppressing the expression of ecdysone-regulated genes in female mosquitoes.

Conclusion: The data suggest that ecdysteroid titers are correlated with Vg synthesis, and methoprene affects vitellogenesis by modulating ecdysteroid action in A. gambiae.

Figure 1. Whole body ecdysteroid titers and the expression of vitellogenin gene (AgVg) of Anophelese gambiae before and after a blood meal

(A). Whole body ecdysteroid titers of A. gambiae. Ecdysteroid levels were estimated using enzyme immunoassay as previously described –. Each time point represents mean ± SE of 5–10 individual insects. Means with the same letter are not significantly different (α ≤ 0.05; ANOVA). (B). The expression of AgVg of A. gambiae. mRNA abundance of AgVg in the fat body determined by quantitative real-time reverse-transcriptase PCR (qRT-PCR). Total RNA was extracted from pools of five fat bodies for each time point. The Y-axis denotes expression levels normalized using AgS7RP levels as an internal control. Mean ± SE of three replications are shown. Means with the same letter are not significantly different (α ≤ 0.05; ANOVA). PE4D: four days after eclosion. PBM: hours after blood meal.

Figure 2. The expression of ecdysone-regulated genes of Anopheles gambiae before and after a blood meal

mRNA abundances of AgEcR (A), AgUSP (B) and AgHR3 (C) in the fat body collected at different stages. Total RNA was extracted from pools of five fat bodies for each time point. The Y-axis denotes expression levels normalized using AgS7RP levels as an internal control. Mean ± SE of three replications are shown. Means with the same letter are not significantly different (α ≤ 0.05; ANOVA). PBM: hours after blood meal.

Figure 3. Primary follicle length at different developmental stages of Anopheles gambiae

Ovaries collected from 6–8 females were used for follicle length measurement. Means with the same letter are not significantly different (α ≤ 0.05; ANOVA). PBM: hours after blood meal.

Figure 4. Effect of methoprene treatment on female reproduction of Anopheles gambiae

Ovaries dissected at 48 h PBM from cyclohexane treated (A), 1 ng methoprene treated (B), 10 ng methoprene treated (C), 50 ng methoprene treated females (D). (OV: ovary; MG: midgut; MT: malpighian tubules). Scale bar: 300 µm. (E) Primary follicle length from methoprene-treated females at 48 h PBM. Mean ± SE of follicles from 6–8 females are shown. Means with the same letter are not significantly different (α ≤ 0.05; ANOVA).

Figure 5. Effects of methoprene treatment on the expression of ecdysone-regulated genes of Anopheles gambiae at 18 h PBM

mRNA abundances of AgEcR (A), AgUSP (B), AgHR3 (C) and AgVg (D) in the fat body collected from cyclohexane and 10 ng methoprene treated female mosquitoes. Total RNA was extracted from pools of five fat bodies for each time point. The Y-axis denotes expression levels normalized using AgS7RP levels as an internal control. Mean ± SE of three replications are shown. Asterisk indicates significant differences between two treatments (α ≤ 0.05; Student’s t-Test).

Figure 6. Proposed model for methoprene action on female reproduction of Anopheles gambiae