Adults of A. chinensis in diapause were collected from Bijie City, Guizhou Province, southwest China. Individuals were kept at 28 ± 1°C with 85 ± 5% humidity under a 16/8 h (light/dark) photoperiod condition (Guo et al., 2019). Mating was used as the standard of diapause termination.

The method of extracting juvenile hormone Ⅲ through high-performance liquid chromatography (HPLC) was referenced Hao et al. (2012). The samples of two A. chinensis (diapause or diapause termination) were placed in a glass homogenizer, 1 ml methanol and 1 ml ethyl ether was added to the homogenate. After homogenization, 4 ml n-hexane was added. It was then centrifuged at 12,000 r/min for 10 min and supernatant was collected. The precipitation was repeatedly extracted with n-hexane 3 times. The collected liquid samples were dried with a nitrogen blower before placing them into the instrument. The volume was made to 1 ml with the mobile phase. The chromatographic conditions include mobile phase methanol: ether (80: 20), column temperature is 25°C, the flow rate is 0.5 ml/min, sample injected 10 μL, ultraviolet detection wavelength is 218 nm; JHⅢ used isocratic elution, the sample was compared with the prototype of the peak area of quantitative. Each treatment had 3 biological replicates.

The under-wing injection method was used. Exogenous JHIII was diluted with acetone to 5, 10, 50, 100, and 200 ng/μL, acetone as a control. For each adult individual, 2 μL acetone was injected by using a 5 μL microsyringe (Shanghai gaoge industrial and trading co., LTD.). We set five repetitions, each with 10 A. chinensis (5 males and 5 females). After injecting, the treated diapause adults were placed in labeled milk tea cups, respectively, in an environment at a temperature of 28 ± 1°C, relative humidity of 85 ± 5%, photoperiod (16 L: 8 D), and the pumpkin (Cucurbita moschata Duch) seedlings were used for rearing (Wu et al., 2019). The number of deaths and the termination of diapause were monitored daily.

Primers (Table 1) used for dsRNA were designed by Primer 5 program. JHEH or JHAMT double-stranded RNA (dsRNA) was prepared using the T7 RNA polymerases containing the appropriate gene and injected into A. chinensis adult. Adults received 1 μL of dsRNA at a concentration of 400 ng/μL in solution; control insects were injected with GFP dsRNA derived from Aequorea victoria genes.

Randomly selected A. chinensis was treated by dsRNA. The reproductive system was dissected in the precooled 75% alcohol from three females and three males every 7 days until mating to investigate tissue-specific expression profiles under a stereomicroscope (SMZ250, Nikon). Day 0 was the control group. We set three repetitions and carefully measured and recorded the size of the reproductive organs.

Fat bodies were dissected in the precooled phosphate buffer saline, and the adherent fat bodies were carefully removed with forceps as thoroughly as possible under a stereomicroscope (SZM-7045, Nikon). The fat bodies from different time periods (every 7 days) were washed twice with 1× PBS buffer fixed with 4% paraformaldehyde for 30 min at room temperature. It was then washed twice with 1× PBS buffer. The fat bodies were then incubated in Nile Red solution (1 mg/ml Nile Red was diluted to 1 ug/mL by 0.1% Triton X-100) for 90 min at room temperature to stain the lipid droplets. Then it was washed twice with 1× PBS buffer. The cell nuclei were stained with 1 μg/ml DAPI (Sigma, D8417, United States) for an additional 15 min. Finally, it was washed twice with 1× PBS buffer. Fat body cell images were captured using a Nikon bright-field inverted light microscope (Tokyo, Japan). Day 0 was the control group. We set three repetitions, each with six A. chinensis (three males and three females).

The total TG levels were determined by the liquid triglycerides (GPO-PAP) method (Liu et al., 2016) using the Triglycerides Assay Kit (Nanjing Jiancheng Institute, China) according to the manufacturer’s instructions. After accurately weighing the fat body, we added anhydrous ethanol at a weight (g) to volume (ml) ratio of 1: 9. Mechanical homogenization was conducted under ice bath condition. Then, after centrifugation at 2,500 rpm for 10 min, the supernatant was taken; 2.5 μL of the supernatant and 250 μL reaction solution were added to a 96-well plate and incubated at 37°C for 10 min. The absorbance was measured at 510 nm using an automated microplate reader (Bio-Rad, Hercules, CA, United States). Three independent biological replicates and five technical replicates were performed for every treatment. The formula used for calculation was as follows.

Total RNAs from whole bodies were isolated using RNAeasy™ Animal RNA isolation Kit with Spin Column (Shanghai, Beyotime) according to the manufacturer’s instructions. RNA concentration was determined using a spectrophotometer (ND-2000, United States, Thermo Scientific). To avoid genomic DNA contamination, total RNA was treated with RNase-free DNase water (Pekin, Solarbio). The first-strand cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit according to the manufacturer’s instructions. Primers (Table 2) used for qPCR were designed by the Primer 5 program. qPCR was performed with a T100TM PCR instrument (T100TM, United States, Bio-Rad) using the iTaqTM Universal SYBR®Green Supermix under the following reaction conditions: 95°C for 10 min; 40 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 20 s. The stable reference gene beta-actin of A. chinensis was used for normalization (Wu 2019). All qPCR reactions were carried out in three biological replicates, and three technical replicates were performed for each sample. The relative expression levels were calculated using the 2^−ΔΔCT method (Livak and Schmittgen 2001).

Results were presented as mean ± SE (standard error) based on at least three independent biological replications. Differences between the two groups were analyzed by an independent t-test. One-way ANOVA followed by Duncan’s multiple comparison was used for the comparison among more than two different conditions. p values less than 0.05 (*), 0.01 (**), or 0.001 (***) were considered to be statistically significant. All data were subjected to statistical analysis using the SPSS software (version 22; SPSS Inc., Chicago, IL, United States). Graphical representations were performed using Excel 2007 software.

High-performance liquid chromatography was used to detect juvenile hormones. The result indicated that the UV absorption peak of standard JHIII was at 218 nm, and the retention time was 8 min approximately (Figure 1A). Subsequently, the JHⅢ of A. chinensis in diapause and diapause termination were detected. A small peak appeared at the time close to the standard sample both in diapause (Figure 1B) and diapause termination samples (Figure 1C). However, the peak in the chromatogram of diapause termination was higher than that in diapause. The JHIII content was quantified according to the peak areas of samples and standards, and the data showed that JHIII concentration in diapause (4.3016 ± 0.0449 ng/μL) was significantly lower than that in diapause termination (9.6873 ± 1.6157 ng/μL) (Figure 1D). It implies that the increase in JHIII content in A. chinensis was involved in terminating diapause.

To further explore the relationship between JH and A. chinensis diapause, the exogenous JHIII injecting was employed. The survival rate of A. chinensis was above 90% after injecting with different JHIII concentrations (Supplementary Table S1). In addition, the rate of mating was counted at day 30. The results showed that JHⅢ injection could increase the rate of mating significantly, and 50 ng/μL JHIII injection had the highest mating rate (91.600% ± 0.038%) (Table 3).

In the male adult, the reproductive system is mainly composed of six parts: a pair of testis (which is divided into seven testicular follicles), a pair of vas deferens, a seminal vesicle, an ejaculatory bulb, an ejaculatory duct, and a pair of accessory glands (Supplementary Figure S1). Correspondingly, in the female adult of A. Chinensis, the reproductive system is mainly composed of a pair of ovaries (which divided into seven ovarioles and with a terminal filament at the apex of each ovariole), a pair of the lateral oviduct, a common oviduct, and a pear-shaped spermatheca, with no accessory glands (Supplementary Figure S2). However, after injection of exogenous JHIII, the male (Figure 2) and female (Figure 3) reproductive organs of A. Chinensis developed rapidly to maturity in about 30 days, and the length and width of reproductive system structures (testis, vas deferens, seminal vesicle, ejaculatory bulb in male; ovarioles, lateral oviducts, common oviduct, spermatheca in female) increased significantly.

The lipid droplets were consumed as energy for reproductive development after diapause termination. Thus, Nile Red staining was used to detect the change of lipid droplets after JHIII injection. In the beginning, the lipid droplets filled the whole fat body. After JHIII injection, the stored lipid droplets in A. chinensis were reduced over time (Figure 4A). In addition, the triglycerides levels were reduced after JHIII injection which is consistent with the results of Nile Red staining (Figure 4B).

After the exogenous JHIII was injected into diapause adults of A. chinensis, the relative expressions of JHEH, JHAMT, and FAS were measured by qPCR. The data showed that the expression of the JHAMT was upregulated significantly. Whereas the expressions of JHEH and FAS were downregulated significantly (Figure 5).

The results given above demonstrated that JH could promote reproductive development but reduce lipid storage suggesting that JH has opposite effects on reproductive diapause.

Firstly, we synthesized the dsRNA of JHEH and JHAMT genes, and the expressions of JHEH and JHAMT in A. chinensis were significantly inhibited after injection of dsRNA (Supplementary Figure S3). The inhibition rate was more than 80%, indicating that the interference effect is better. Next, the anatomy of the reproductive system of the A. chinensis after dsRNA injection is shown in Figures 6, 7. Compared with the control group (CK or dsGFP), the male or female reproductive system of the dsJHAMT experimental group developed significantly slower, while the dsJHEH experimental group developed faster. With the passage of time, the testis, vas deferens, seminal vesicles and ejaculatory bulb gradually developed in the dsJHEH group developed and matured earlier, while the dsJHAMT group developed more slowly (Supplementary Figure S4). The ovarioles, lateral oviduct, spermatheca, and common oviduct in each experimental group developed gradually over time, and the dsJHAMT group developed slowly at 0, 7, 14, 21, 28, 35, and 42 days, while the dsJHEH group developed on the 42nd day (Supplementary Figure S5).

The results of Nile Red staining showed that the fat body cells in the early stage of diapause were full of lipids, and the fat was gradually consumed over time. Compared with the CK and dsGFP group, the fat consumption of dsJHEH was significantly higher, while the fat consumption of dsJHAMT was not obvious (Figure 8). Compared with the control group (dsGFP) and the blank group (CK), the consumption rate of triglycerides in the dsJHAMT group was relatively slow, while the consumption of triglycerides in the dsJHEH group was intensified (Figure 9A), which was consistent with the results of Nile Red staining.

We calculated the concentration of juvenile hormone according to the standard curve (Supplementary Figure S6). Juvenile hormones in diapause A. chinensis are a process of accumulation (CK group in Figure 9B). In the dsGFP group, the concentration of juvenile hormone increased from 140.04 ng/g to 414.38 ng/g with time. After injection of dsJHEH, the degradation of juvenile hormone was inhibited, and the growth rate of juvenile hormone concentration was significantly higher than that of other groups. Compared with dsGFP, after injection of dsJHAMT, the synthesis of juvenile hormone was significantly inhibited, and the growth rate of juvenile hormone was slowed down (Figure 9B and Supplementary Table S2).

At last, the mean mating occurrence time after dsRNA injection was calculated. The results showed that the mean mating occurrence times of the CK and dsGFP groups were at 58.67 ± 0.88 and 60.33 ± 0.88 days, respectively. The injection of dsJHAMT was at 77.67 ± 1.20 days, which was delayed significantly compared with other groups, while the dsJHEH injection group was at 52.00 ± 0.58 days, and their mating occurrence time was significantly earlier (Figure 9C).