Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes

Abstract

Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher's Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.

Fig 1. Follicular development in PPF-treated mosquitoes.

(A) Ovarian follicles were collected at the indicated time points after PPF treatment. After fixation, follicles were stained with DAPI. Images were captured using a Zeiss LSM 880 confocal microscope. Scale bars represent 50 μm. (B) Differential interference contrast (DIC) images of ovarian follicles after PPF treatment. PE, Post eclosion; PBM, Post blood-meal. 1°, primary follicle; 2°, secondary follicle; G, germarium; NC, nurse cell.

Fig 2. PPF exposure affected the levels of Glycogen and TAG.

Female mosquitoes were treated with PPF (70 μg/cm²) at 72 h PE. (A) Quantitative measurement of glycogen in the whole-body mosquitoes at 120 h PE and 24 h PBM. The levels of glycogen were normalized to the protein contents of the samples. The relative abundance of the untreated control is set at 1. Error bars represent the standard deviation (SD) of three biological replicates. The differences between the PPF-treated and cyclohexane-treated mosquitoes were analyzed using paired t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (B) Glycogen in the fat body was visualized after PAS staining. The intensity of the pink/ purple color reflects the levels of glycogen. (C) Quantitative measurement of TAG in the whole-body mosquitoes. The levels of TAG were normalized to the protein contents of the samples. (D) Nile red lipid staining of lipid droplets in the fat body. Scale bar, 50 μm. PE, Post eclosion; PBM, Post blood-meal.

Fig 3. PPF treatment altered the expression of metabolic genes.

Female mosquitoes were treated with PPF at 72 h PE. Quantitative RT-PCR assays were performed to compare the expression of selected genes involved in carbohydrate metabolism (CM) and lipid metabolism (LM). mRNA levels were measured in the fat body at 120 h PE and 24 h PBM. Data are presented as mean ± SD from three independent replicates. Statistical analyses were performed using paired t-test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig 4. Expression patterns of selected hormone-regulated genes in PPF-treated mosquitoes.

The mRNA levels were analyzed in the fat body of female mosquitoes at the indicated time points after PPF exposure at 72 h PE. The cyclohexane-treated mosquitoes were used as a control group. Transcript abundance was normalized to that of the Ribosomal Protein S7 (RpS7) gene. Data are shown as the mean ± standard deviation of three biological replicates. Statistical analysis was performed using a Student’s t-test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig 5. Effect of Met depletion on the PPF-triggered alterations of gene expression.

DsMet was injected into adult female mosquitoes within 1 h after eclosion. Untreated and dsGFP-injected mosquitoes were used as control groups. At 72 h PE, the Met RNAi mosquitoes and control groups were treated with cyclohexane (Cyc) or PPF (70 μg/cm²). Fat bodies were collected at 96 h PE (A) or 24 h PBM (B). Relative mRNA levels of the indicated genes were determined using real-time PCR. Error bars represent the standard deviation of three replicates. Statistical significance was determined using a Student’s t-test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig 6. Implication of Met in the impaired egg maturation in the PPF-treated female mosquitoes.

Adult female mosquitoes were injected with dsRNAs within 1 h after eclosion. The injected and uninjected mosquitoes were treated with cyclohexane (Cyc) or PPF at 72 h PE. All the mosquitoes were given a blood meal at 120 h PE. (A) Length of primary follicles. Follicular lengths (N = 30 follicles) were measures at 120 h PE and 48 h PBM. (B) Egg shape parameter (egg L/W ratio) was calculated after measuring the lengths and widths of 30 eggs. (C) Average number of eggs produced by each female mosquito. Oviposited eggs were counted at 6 days PBM. Data are shown as the mean ± standard deviation of three biological replicates (10 mosquitoes/replicate). (D) Hatching rates for different treatment groups. Eggs were allowed to hatch for 7 days to record the hatching rate. Error bars illustrate the standard deviation of three biological replicates (10 mosquitoes/replicate). Statistical analysis was conducted using paired t-test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (E) Ovarian follicular development. Follicles were dissected at 120 h PE and 48 h PBM. Differential Interference Contrast (DIC) images were taken using Zeiss Axiocam ERc5. PE: Post eclosion, PBM: Post blood-meal, 1°: primary follicle, 2°: secondary follicle, G: germarium.