The Amino Acid-Mediated TOR Pathway Regulates Reproductive Potential and Population Growth in Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae)

The predatory mirid bug, Cyrtorhinus lividipennis Reuter, feeds on brown planthopper (BPH) eggs that are deposited on rice and gramineous plants surrounding rice fields. The development and reproduction of C. lividipennis are inhibited by feeding on BPH eggs from gramineous species, and the underlining regulatory mechanism for this phenomenon is unclear. In the present study, HPLC-MS/MS analysis revealed that the concentrations of six amino acids (AAs:Ala, Arg, Ser, Lys, Thr, and Pro) were significantly higher in rice than in five gramineous species. When C. lividipennis fed on gramineous plants with BPH eggs, expression of several genes in the target of rapamycin (TOR) pathway (Rheb, TOR, and S6K) were significantly lower than that in the insects fed on rice plants with BPH eggs. Treatment of C. lividipennis females with rapamycin, dsRheb, dsTOR, or dsS6K caused a decrease in Rheb, TOR, and S6K expression, and these effects were partially rescued by the juvenile hormone (JH) analog, methoprene. Dietary dsTOR treatment significantly influenced a number of physiological parameters and resulted in impaired predatory capacity, fecundity, and population growth. This study indicates that these six AAs play an important role in the mediated-TOR pathway, which in turn regulates vitellogenin (Vg) synthesis, reproduction, and population growth in C. lividipennis.

Frontiers in Physiology | www.frontiersin.org 2 November 2020 | Volume 11 | Article 617237 The feeding and reproduction of insect species are closely correlated with nutritional factors (Yu et al., 1996). Cyrtorhinus lividipennis is an unusual species that exhibits both herbivorous and predatory feeding habits (Bentur and Kalode, 1985). When C. lividipennis consumed a diet of planthoppers that inhabited gramineous species such as Echinochloa glabrescens, Leptochloa chinesis, Digitaria ciliaris, Cyodon dactylon, and Eleusine indica growing on the bunds surrounding rice fields, their development and reproduction were reduced in comparison to those consuming planthoppers (such as BPH eggs) on rice plants (Pomari-Fernandes et al., 2015), indicating that nutrition from rice plant is beneficial to the predator. When C. lividipennis was fed on eggs of the rice meal moth, Corcyra cephalonica (Lepidoptera: Pyralidae), the predator successfully reached the adult stage (Murugan and George, 1992). Similarly, Telenomus remus (Hymenoptera:Platygastridae) showed normal development when fed on C. cephalonica eggs, suggesting that C. cephalonica is a promising factitious host (Matteson, 2000). Collectively, these results indicate that C. cephalonica eggs or planthopper eggs/nymphs from rice plants provide adequate nutrients for successfully rearing predatory insects.
Predation capacity is influenced by changes in the environment, host morphology, and nutrient composition (Wakde, 1995;Laba and Heong, 1996;Cortesero et al., 2000). With respect to the latter category, proteins, free amino acids (AAs), sugars, lipids, inorganic salts, and vitamins provide energy and compounds needed for insect growth and development (Shah et al., 2005). AAs are among the most important nutrients and participate in various metabolic processes in insects (Sogawa, 1972); furthermore, AAs were shown to significantly impact the balance between fecundity and lifespan in Drosophila (Grandison et al., 2009). Our recent transcriptomic analysis revealed that, when newly-emerged C. lividipennis fed on BPH eggs from any of the five gramineous species mentioned above, the expression levels of the TOR and S6K genes in the target of rapamycin (TOR) pathway were significantly downregulated, compared to those feeding on BPH eggs from rice plants or C. cephalonica eggs (data not shown).
The TOR signaling pathway transduces nutritional signals that stimulate vitellogenin (Vg) synthesis in insect fat body, which subsequently activates egg development (Kim and Guan, 2011;Roy and Rikhel, 2011;Perex-Hedo et al., 2013). In Aedes aegypti, the TOR signaling pathway genes (e.g., S6K, Rheb, and TSC2) relay nutritional information during egg development (Hansen et al., 2004Roy and Rikhel, 2011). Furthermore, the knockdown of TOR gene in A. aegypti reduced Vg transcription and resulted in reduced numbers of deposited eggs, suggesting that the TOR pathway is a critical component in Vg biosynthesis and the maturation of oocytes (Hansen et al., 2004Roy and Rikhel, 2011). In Blattella germanica, the TOR pathway reportedly links nutritional signals with juvenile hormone (JH) and Vg synthesis (Maestro et al., 2009). An analogous relationship between the TOR signaling pathway and JH synthesis was identified in BPH (Lu et al., 2016). Collectively, these studies indicate that Vg transcription and egg maturation are controlled by endocrine hormones and the TOR signaling pathway in insects.
In the present study, we explore the question of why a diet of BPH eggs from gramineous plants is an unsuitable food source for C. lividipennis. This study illuminates one of the underlying regulatory mechanisms of this phenomenon and provides valuable information regarding the role of six AAs in the reproduction of C. lividipennis.

Plant Materials and Insects
The BPH-susceptible rice cultivar Ningjing 4 was used in this study. Seeds were planted in a cement pool, and seedlings were transferred into plastic pots and arranged in hills at the six-leaf stage as previously described Ge et al., 2020a). All experiments utilized rice at the tillering stage (40 days).
The seeds of gramineous plants, including L. chinesis, D. ciliaris, C. dactylon, and E. indica, were purchased from Jiangsu Leerda Seed Industry Co., Ltd. (LiYang, Jiangsu). Seeds of the barnyard grass E. glabrescens were collected from rice fields located at the Yangzhou University Farm. Seeds were sown in plastic pots (16 cm diameter × 15 cm high). The six gramineous plants were used in experiments 40 days after germination because plenty of BPH eggs were laid on these plants at this time. Each newly-emerged C. lividipennis female was offered 10 rice plants with BPH eggs or 50 C. cephalonica eggs as food sources.
The BPH strain utilized herein was provided by the China National Rice Research Institute (Hangzhou) and reared as described previously (Ge et al., 2020a). Cyrtorhinus lividipennis was obtained from rice plants cultivated in Yangzhou and maintained on Ningjing 4 rice containing N. lugens eggs or nymphs.

Reagents and Quantitation of Free Amino Acids
Amino acids, vitamins, inorganic salts, and other reagents were purchased from Sinopharma Chemical Reagent Co. Ltd. (Shanghai, China). Stems (0.1 g fresh weight) were removed from rice (tillering stage) and five gramineous species at 40 days after germination, respectively, and transferred to 1 ml of 0.01 N HCl. Suspensions were allowed to settle for 15 min at ambient temperature, and a 0.5 ml volume was centrifuged at 10,000 × g. AAs were extracted using the EZ:Fast Free AA Kit (Phenomenex, CA, United States). HPLC-MS/MS analysis of free AAs in extracted samples was conducted as described (Florencio-Ortiz et al., 2018). AA standards were supplied with the EZ:Fast kit, and calibration curves were calculated for each AA. Data were analyzed with Agilent 5975 software.
Artificial Diets for C. lividipennis Artificial diets were formulated as described previously (Ge et al., 2020b). Compounds were dissolved in doubledistilled water (ddH 2 O), and the pH was adjusted to 6.4 with 4% KOH in a total volume of 100 ml. Diets were Frontiers in Physiology | www.frontiersin.org prepared with AAs (+6 AAs:Ala, Arg, Lys, Ser, Pro, and Thr), without AAs (−6 AAs:−Ala, −Arg, −Lys, −Ser, −Pro, and −Thr), or lacking individual AAs (−1 AA:−Ala, −Arg, −Lys, −Ser, −Pro, or −Thr). Glass cylinders (7.5 × 1.2 cm) were used as feeding chambers. A 50 μl aliquot of the artificial diet was sandwiched between layers of a Parafilm membrane as described previously (Ge et al., 2020a). The diet capsule was replaced on alternate days, and 200 μl ddH 2 O was added daily to maintain RH (He et al., 2014). A black cotton cloth was used to exclude light from the cylinders; however, the open end containing the diet capsule was left uncovered to provide light. Cyrtorhinus lividipennis feeding (n = 10 first instar individuals) was facilitated by puncturing the inner Parafilm membrane, and 10 chambers were used to rear nymphs in each treatment. Rearing was conducted in growth chambers maintained at 26 ± 2°C, 80% RH with a 16L:8D photoperiod. Mortality was recorded on alternate days.

dsRNA Preparation and Microinjection
Rheb, TOR, and S6K were amplified from cDNA using primer sets Rheb-F/Rheb-R, TOR-F/TOR-R, and S6K-F/S6K-R (Supplementary Table S1). The green fluorescent protein (GFP) gene encoding GFP was used as a negative control; this was amplified as a 688-bp fragment from pHT3AG with primers GFP-F and GFP-R. The reaction conditions were as follows: 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a final extension at 72°C for 10 min. Cloned PCR products were used as templates to re-amplify the genes for dsRNA synthesis using the following primer sets: Rheb-T 7 F/Rheb-T 7 R, TOR-T 7 F/TOR-T 7 R, S6K-T 7 F/S6K-R, and GFP-T 7 F/GFP-T 7 R (Supplementary Table S1). A DNA gel purification kit (Omega Bio-tek, Doraville, GA, United States) was used to purify PCR products. The T 7 RiboMAX ™ Express RNAi System (Promega, Madison, WI, United States) was used to generate dsRNAs by in vitro transcription. The dsRNA products were dissolved in diethyl pyrocarbonate (DEPC)-treated water at 5 μg/μl and stored at −80°C.
Fifth instar C. lividipennis nymphs were anesthetized with CO 2 prior to dsRNA injection (Lin et al., 2016). Injection of the intra-thoracic region was carried out using a Nanoject II microinjection device (Drummond Scientific, Broomall, PA, United States; Liu et al., 2010). dsRNA (50 ng in 50 μl) was injected into fifth instar nymphs; after a 2 h recovery period, nymphs were allowed to feed on the complete artificial diet. Insects were collected at 2 days after emergence (DAE) and used for total RNA extraction.

Methoprene and Rapamycin Treatments
A stock solution (100 ng/nl) of the JH analog (JHA) methoprene (Sigma-Aldrich, St. Louis, MO, United States) was prepared in acetone as described (Ge et al., 2020a) and diluted to 10 μg/μl in acetone and ddH 2 O (1:10 v/v). Fifty nanoliters of 1.0 ng/nl JHA solution was topically applied to the dorsal side of newly emerged female as described (Ge et al., 2020a). Methoprene-treated C. lividipennis females were transferred into glass cylinders and maintained on normal artificial diets.
C. lividipennis females were collected 48 h after topical application; quantitative real-time PCR (qPCR) was used to evaluate the expression of Rheb, TOR, S6K, and Vg, and immunoblotting was used to detect phosphorylation level of S6K and Vg. Treatments and controls consisted of three independent biological replicates.
Rapamycin (Sigma-Aldrich St. Louis, MO, United States) was dissolved in ethanol (Lu et al., 2016). The abdominal region of fifth instar nymphs was injected with a 100 nl solution of 2.0 nM rapamycin, whereas negative controls were injected with an equal volume of ethanol. The nymphs were supplied with the complete artificial diet containing AAs and reared until adults emerged. Newly emerged females were collected and treated with methoprene. The expression of Rheb, TOR, S6K, and Vg were determined using qPCR, and the phosphorylation of S6K and Vg were determined by western blot (WB) analysis 48 h after the topical application of JHA methoprene. Treatments and controls consisted of three independent biological replicates, with 15 females for each replicate.

Quantitative Real-Time PCR
Trizol reagent (Invitrogen, Carlsbad, CA, United States) was used to extract RNA from C. lividipennis. cDNA was synthesized with PrimeScript RT Reagent Kit and gDNA Eraser (TakaRa Beijing, China). cDNA was synthesized in 20 μl reaction volumes at 37°C for 15 min in a mixture containing random hexamers and oligo dT primers.

Protein Extraction and Determination
Soluble proteins were extracted from ovaries and fat bodies of 50 dsTOR-treated females and 50 dsGFP-treated control females at 2 DAE as described by Ge et al. (2010). The Bradford method (Bradford, 1976) was used to determine protein concentrations. A 595 values were measured by UV spectrophotometry, and protein content was determined with a standard curve of BSA (Shanghai Biochemistry Research Institute, Shanghai, China). Treatments and controls consisted of three independent biological replicates.

Female Body Mass and Isolation of Ovaries
The weights of 10 dsTOR-and 10 dsGFP-treated females (control) were recorded at 2 DAE. Treatments and controls consisted of three independent biological replicates (n = 10, 10 BPH females for each replicate, N = 3, 3 replicates).

JH III and Ecdysteroid Titers
Titers of JH III and ecdysteroid were measured in adult females by HPLC/MS (Cornette et al., 2008;Giebbultowicz et al., 2008). 20-hydroxyecdysone and JH III standards were obtained from Sigma (St. Louis, MO, United States). Treatments and controls were replicated three times with five females for each replicate.

Measurement of Predation Capacity in C. lividipennis
The predation capacity of C. lividipennis was measured as described by Base et al. (2002) with minor modifications. First instar nymphs of the prey, N. lugens (n = 20), were placed in glass tubes (3 × 25 cm) containing four 15 days-old rice seedlings of rice cv. "Ningjing 4, " which is susceptible to N. lugens. Newly emerged (<24 h) C. lividipennis adult females were individually placed into glass tubes, which were then sealed with nylon mesh. Surviving N. lugens nymphs were recorded daily, and the predation number per C. lividipennis was assessed based on survival counts of N. lugens nymphs at days 1 and 3. Treatments and controls consisted of five independent biological replicates.

Western Blot Analysis
Immunoblotting was conducted as described previously with minor modifications (Ge et al., 2019). Fat bodies and ovaries were separately homogenized in lysis buffer (0.5 ml) supplemented with protease and phosphatase inhibitors (Lu et al., 2016) and incubated for 1 h at 4°C. Lysates were centrifuged and protein concentration in the resulting supernatant was analyzed using the Bradford method as described above. Total 30 μg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes; the membranes were treated with a blocking solution [5% nonfat dry-milk in 10 mM Tris-buffered saline (TBS), pH 7.4, containing 0.5% TBS Tween-20 (TBST)] for 1 h and then incubated with primary antibodies at room temperature for 2 h. Anti-phospho-p70 S6 kinase (p70-Thr-389 S6K) polyclonal antiserum (1:5,000) was obtained from Cell Signaling Technology (Danvers, MA, United States), and anti-Vg antiserum (1:5,000) was prepared by Nanjing Kingsley Biotechnology Co. Ltd. (Nanjing, China). Antiserum to β-actin (1:5,000; Cell signaling Technology, Davers, MA, United States) served as a loading control. Membranes were washed with TTBS three times, 5 min each, and incubated within the TBS buffer containing goat anti-rabbit IgG secondary antibodies-conjugated to horseradish peroxidase (1: 8,000 dilution) for 1 h at room temperature. Reactive proteins were visualized with chemiluminescent substrates with the GBOX-Chemi XT4 system (Syngene, Cambridge, UK) as described previously (Lu et al., 2016).

Population Growth
Two groups were established to monitor population growth: dsGFP-treated females mated with untreated males (control group), and dsTOR-treated females mated with untreated males (treatment group). A randomized complete block containing five replicates was used as an experimental design. Newly emerged C. lividipennis (two pairs) were released on rice plants containing BPH eggs at the tillering stage (40 days after germination) and enclosed in nylon cylindrical cages as described (Ge et al., 2019). When the third instar nymphs of the next generation emerged (~25 days), groups were inspected daily and the third instar nymphs were counted; these nymphs were transferred to new plastic pots with tillering-stage rice plants. Nymphs were examined every 2 days until adults emerged; numbers of both sexes were recorded until the females died. Numbers of adults from the new generation and unhatched egg counts were used to calculate hatch rates and the ratio of adults/ adults + unhatched eggs (Ge et al., 2019). The population growth index (PGI) was expressed by the ratio N1/N0; this was determined by dividing the total number (N1) of adults of next generation + unhatched eggs by the number of adults released (N0 = 8, 4 pairs).

Statistical Analyses
Statistics were obtained with SPPS v. 18.0 (SPSS Inc., Chicago, IL, United States). The Shapiro-Wilk test was utilized to examine normality of data variances, and the t-test was performed to compare means of two variables. Fisher's protected least significant difference (PLSD) test was applied to multiple comparisons of the means. Data points were considered significant at p < 0.05. Values were expressed as means ± SEM.
Cyrtorhinus lividipennis females fed on diets lacking certain AAs led to upregulated TSC1 and TSC2; expression was up by 54 and 55% in Arg-deprived, 27 and 38% in Lys-deprived, 45 and 0% in Ser-deprived (TSC2), and 33 and 32% in Ala-deprived groups as compared to females reared on all AAs, respectively (Figures 2D,E). There were no significant differences in TSC1 or TSC2 expression levels in females feeding on Pro-or Thr-deprived artificial diets (Figures 2D,E). TSC1 expression was not significantly different in the Ser-deprived diet relative to the complete diet ( Figure 2D).
There was a noticeable decrease in S6K phosphorylation when C. lividipennis females were deprived of one or more AAs (Figure 2E).

TOR Pathway Transduces AA Signals to Regulate Vg Synthesis
Vitellogenin expression in C. lividipennis was 27.3-69.1% lower when diets consisted of gramineous plants with BPH eggs, rice plants alone, or no food (starved) as compared to females feeding on BPH eggs from rice plants or C. cephalonica eggs (Figure 3A; F = 144.6, df = 8, 26, p = 0.0001). When C. lividipennis females were fed on diets lacking one or more AAs, Vg expression was down by 31-74% as compared to females fed on a complete artificial diet at 2 DAE ( Figure 3B; F = 220.9, df = 7, 23, p = 0.0001). Western blot analysis confirmed that feeding on multiple or single AA-deprived artificial diets resulted in a significant reduction of Vg protein ( Figure 3F). The efficiency of silencing Rheb, TOR, and S6K was approximately 74.4, 74.2, and 73.1% at 2 DAE, respectively ( Figure 3C). Cyrtorhinus lividipennis females treated with A4, no food source (starvation); A5, Cynodon dactylon with BPH eggs; A6, Digitaria ciliaris with BPH eggs; A7, Eleusine indica with BPH eggs; A8, Echinochloa glabrescens with BPH eggs; and A9, Leptochloa chinensis with BPH eggs. Panels (A-E) show expression of Rheb, TOR, S6K, TSC1, and TSC2 as measured by qPCR 2 days after emergence (DAE); β-actin was used as a reference gene for normalizing the data. Each treatment and control contained three replicates, and error bars represent the mean ± SEM. Columns labeled with different letters indicate a significant difference at p < 0.05 by the Student's t-test.
Rapamycin treatment also reduced Vg expression levels, which were 68% lower than females treated with the ethanol control ( Figure 3E; F = 390.3, df = 1,5, p = 0.0001). Western blot analysis showed that both Vg levels and S6K phosphorylation were reduced in the rapamycin-treated females relative to the ethanol control ( Figure 3N).

TOR Pathway Functions via JH to Regulate Vg Synthesis
When C. lividipennis were fed on gramineous plants with BPH eggs, rice plants without BPH eggs, or no food (starvation), JHAMT expression was down by 28-70% as compared to females feeding on BPH eggs from rice plants or C. cephalonica eggs (Figure 4A; F = 35.4, df = 8,26, p = 0.0001). When C. lividipennis females were deprived of multiple or individual AAs, JHAMT expression levels were down by 20-94% as compared to females feeding on a complete artificial diet (Figure 4B; F = 91.4, df = 7,23, p = 0.0001). Rapamycin treatment significantly The application of methoprene to C. lividipennis females treated with rapamycin, dsTOR, dsRheb, or dsS6K was partially rescued by Vg expression. For example, Vg expression was 117% higher when methoprene was added to rapamycin-treated females as compared to rapamycin alone ( Figure 4E; F = 48.0, df = 2,8, p = 0.0002), upregulation was 86% higher when methoprene was added to dsRheb-treated females (Figure 4F;  s t-test). Panels (G-N) show Vg protein levels and pS6K phosphorylation status in C. lividipennis treated with dsRheb, dsTOR, dsS6K, and rapamycin. Proteins were detected with anti-Vg or anti-pS6K antisera and visualized by western blot analysis. Antiserum to β-actin was used as a loading control.

DISCUSSION
Nutrients play a critical role in regulating insect reproduction . Earlier studies demonstrated that a diet of gramineous plants containing eggs of BPH resulted in reduced development and reproduction in C. lividipennis compared to insects feeding on BPH eggs from rice plants or C. cephalonica eggs (Yu et al., 1996). However, the regulatory mechanism of this phenomenon was unclear. The present study shows that rice plants contain higher concentrations of Ala, Lys, Pro, Arg, Ser, and Thr than the five gramineous species ( Table 1), suggesting that these six AAs might be associated with development and reproduction of C. lividipennis. We also found that TOR was significantly upregulated when C. lividipennis was supplied rice plants containing BPH eggs as compared to gramineous species with eggs based on transcriptomic data (data not shown). Using a defined artificial diet (Ge et al., 2020b), we infer that the TOR signaling pathway senses these six AA signals and regulates downstream genes and proteins, thus leading to an increase in Vg synthesis, reproduction, and population growth of C. lividipennis. Target of rapamycin is a serine/threonine kinase that consolidates nutritional signals via AAs (Kim and Guan, 2011). The disruption of the TOR signaling pathway has been associated with the reduced reproductive capacity that occurred when nutrients were limiting in Tribolium castaneum, B. germanica, and A. aegypti (Hansen et al., 2004Parhasarathy and Palli, 2011). When C. lividipennis was supplied with a diet of gramineous plants plus BPH eggs or rice plants alone, multiple genes in the TOR pathway were repressed (Figure 1), and similar results were obtained with diets lacking one or multiple AAs (Figure 2). The TOR signaling pathway regulates multiple cellular functions including proliferation, apoptosis, growth, and autophagy by integrating a multitude of nutritional signals Histogram shows means ± SEM, and columns labeled with different lowercase letters were significantly difference at p < 0.05 using the Student's t-test. Each treatment and control were from 15 independent biological replicates. Structural changes were shown in the ovaries of C. lividipennis females subjected to silencing of TOR pathway-related genes at 7 DAE. Representative images from dsGFP-treated control females (D); and dsRheb-(E), dsTOR-(F), and dsS6K-(G) treated females. Reproductive tracts were dissected from at least 10 females from each group and photographed with a Leica DMR connected to a Fuji FinePix S2 Pro digital camera (Tokyo, Japan). Scale bar, 500 μm. Frontiers in Physiology | www.frontiersin.org (Yang et al., 2006). The tumor suppressors, TSC1 and TSC2, function in both Drosophila and mammals as negative regulators of Rheb (Fingar and Blenis, 2004;Pan et al., 2004;Yang et al., 2006). TORC1, which is sensitive to rapamycin, modulates cellular growth and protein biogenesis by phosphorylating the translational regulators 4EBP1 and S6K (Kwiatkowski, 2003). Previous research demonstrated that AA-dependent nutritional signaling mediates S6K phosphorylation in the TOR signaling pathway (Maestro et al., 2009). In this study, RNAi-mediated knockdown of Rheb, TOR, and S6K led to repressed expression of Vg and reduced Vg synthesis and a decline in S6K phosphorylation (Figure 3). Similarly, the TOR inhibitor rapamycin also led to reduced levels of Vg and S6K phosphorylation in C. lividipennis at 2 DAE (Figure 3). Target of rapamycin is known to transduce nutritional signals in many organisms including vertebrates, invertebrates, and yeast (Wang and Pround, 2009;González and Hall, 2017). In the red flour beetle, T. castaneum, the two primary regulators of Vg transcription were JH and nutrition (Parthasarathy et al., 2010;Sheng et al., 2011). With respect to JH, we showed that JHAMT Frontiers in Physiology | www.frontiersin.org expression was reduced when C. lividipennis was fed on the gramineous plants containing N. lugens eggs or supplied with diets lacking one or more of six identified AAs. RNAi-mediated knockdown of the TOR pathway genes (Rheb, TOR, and S6K) or injection with the TOR inhibitor rapamycin also resulted in a dramatic reduction in JHAMT expression (Figure 4). Thus, the TOR signaling pathway in C. lividipennis regulated JH biosynthesis in response to AA signals. Furthermore, the application of the JHA methoprene to C. lividipennis females injected with rapamycin or treated with dsRheb, dsTOR, or dsS6K partially rescued Vg, Rheb, TOR and S6K expression, Vg levels, and S6K phosphorylation (Figures 4, 5). Therefore, we inferred that AA signals were transduced through the TOR pathway and regulated Vg synthesis through influencing JH biosynthesis in C. lividipennis. In female insects, JH participates in the regulation of vitellogenesis (Fronstin and Hatle, 2008;Shiao et al., 2008), and the regulation of JH via nutritional signals and the TOR pathway is well-established for several insect species (Tu et al., 2005;Sheng et al., 2011;Perex-Hedo et al., 2013).
Target of rapamycin has a critical function in the development of insect reproductive systems (Park et al., 2006). The synthesis of Vg and its uptake by oocytes play a critical function in the invertebrate reproductive process (Tufail and Takeda, 2009). Results in our study demonstrate that dietary dsTOR causes a dramatic reduction in soluble proteins in ovaries and fat bodies, increases ecdysteroid titers, and decreases JH titers, female longevity, body weight, predatory capacity, number of laid eggs, and the oviposition period (Supplementary Figure S1 and Figure 6). Furthermore, RNAi-mediated knockdown of TOR pathway genes interfered with normal development of the female reproductive system and the uptake of Vg by oocytes (Figures 6, 7). dsTOR treatments also led to reduced numbers of offspring, declined hatching rates, and reduced PGI ( Table 2), which is consistent with TOR function in tissue development, cell growth and proliferation, and nutritional signaling (Wullschleger et al., 2006). The process of vitellogenesis in many insects starts with the uptake of AAs and proteins (Hansen et al., 2004;Abrisqueta et al., 2014). The AA/TOR and insulin pathways function as nutritional sensors, impact reproductive organs, and modulate the biosynthesis of ecdysteroids and JH (Smykal and Raikhel, 2015). Ecdysteroids regulate vitellogenesis and the maturation of egg when nutritional resources are adequate (Gaziova et al., 2004;Terashima and Bownes, 2004). Previous studies demonstrated that RNAimediated knockdown of TOR expression in insects can decrease number of eggs laid and can impact ovary development and fecundity Lu et al., 2016;Liu et al., 2017;Zhuo et al., 2017).
In summary, this study shows that elevated concentrations of six AAs in rice plants function as key nutritional signals in JH and ecdysteroids biosynthesis, Vg biogenesis, ovary development, reproduction, and population growth in C. lividipennis (Supplementary Figure S2). The present study provides information on the AAs that foster reproduction of C. lividipennis illustrates the relationship between host nutrition and insect reproduction in predatory mirid bugs. It is important to note that plants produce many other secondary metabolites that could either promote or interfere with metabolism, neural transmission, or reproduction of herbivores (Wink, 1988(Wink, , 2003. Investigations are underway to explore whether secondary metabolites present in gramineous species impact the reproduction and development of C. lividipennis, which are necessary steps in formulating an artificial diet that promotes longevity and fecundity of this important predator.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
LG wrote original draft and designed the experiment. HZ, SZ, and JX perform this experiment. QW performed the data analysis. QS modified the final manuscript. All authors contributed to the article and approved the submitted version.