*4.3. Fecundity Examination*

Twenty adult flies (male:female = 1:1, 24 h post emergence) were placed in vials with or without CPT and the number of eggs laid at the 9th d post treatment was collected and counted. The egg production was measured within 24 h and three independent trials were performed.

#### *4.4. Immunostaining*

After the ovaries of female flies were dissected in PBS, the tissues were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, rinsed with PBT (0.1% Triton X-100 in PBS) three times for 10 min each, blocked in 5% NGS (Normal goat serum) for 1 h, and incubated with primary antibodies overnight. Then, the samples were washed three times with PBT for 10 min each. After incubation with secondary antibody for 3 h, the tissues were stained with Hoechst for 10 min, and washed again with PBT 3 times.

The primary antibodies used in this study were listed as follows: rabbit anti-pMad (1:800; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Caspase 3 (1:2000, Cell Signaling Technology, USA), rabbit anti-pErk (1:400; Cell Signaling Technology, USA) mouse anti-lacZ (1:10,000; abcam, Cambridge, UK), and rabbit anti-LacZ (1:10,000; abcam, UK). Other antibodies were purchased from Temasek Life Sciences Laboratory, including mouse anti-3A9, rabbit anti-α-Spectrin [56], mouse anti-Bam, chicken anti-GFP, rabbit anti-Tkv [32], guinea pig anti-Vasa, mouse anti-Arm, mouse anti-En, rat anti-Fluorescein (FITC), Cy3- and Cy5-goat against rabbit, mouse, chicken, rat and guinea pig secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA), Inc. The DNA dye used was Hoechst 33258 (1:5000; Cell Signaling Technology, USA). Samples were analyzed with an upright confocal microscopy.

For TUNEL (Roche, # 12156792910, Penzberg, Germany), the ovaries were dissected in PBS, and fixed in 4% PFA for 1 h. After washing three times with PBS for 10 min each, the tissues were incubated in Permeabilization solution for 2 min on ice. In total, 5 μL of enzyme solution was added to the 45 μL label solution to obtain 50 μL TUNEL reaction mixture, and this was shaken for 1 h at 37 ◦C, and then we continued with immunostaining procedures as mentioned previously.
