BmCncC/keap1-pathway is involved in high-temperature induced metamorphosis regulation of silkworm, Bombyx mori

The global warming has affected the growth, development and reproduction of insects. However, the molecular mechanism of high temperature stress-mediated metamorphosis regulation of lepidopteran insect has not been elucidated. In this study, the relationship between the insect developmental process and endogenous hormone level was investigated under high temperature (36 ° C) stress in Bombyx mori (B. mori). The results showed that the duration of 5th instar larvae were shortened by 28 ± 2 h, and the content of 20E was up-regulated significantly after 72 h of high temperature treatment, while the transcription levels of 20E response genes E93, Br-C, USP, E75 were up-regulated 1.35, 1.25, 1.28, and 1.27-fold, respectively. The high temperature treatment promoted the phosphorylation level of Akt and the downstream BmCncC/keap1 pathway was activated, the transcription levels of 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12, 1.51, 2.17 and 1.23-fold, respectively. After treatment with double stranded RNA of BmCncC (dsBmCncC) in BmN cells, the transcription levels of cyp302a1 and cyp306a1 were significantly decreased, whereas up-regulated by 2.15 and 1.31-fold, respectively, after treatment with CncC activator Curcumin. These results suggested that BmCncC/keap1-mediated P450 genes (cyp302a1, cyp306a1) expression resulted in the changes of endogenous hormone level, which played an important role in the regulation of metamorphosis under high temperature stress. Studies provide novel clues for understanding the CncC/keap1 pathway-mediated metamorphosis regulation mechanism in insects. Author Summary Mammalian nuclear transcription factor Nrf2 (NF-E2-related factor 2) plays an important role in the stress response of cells. CncC is a homolog of mammalian Nrf2 in insect, regulating the genes expression of insect antioxidant enzymes and cytochrome P450 detoxification enzyme. Evidence suggests that the CncC/Keap1 pathway also plays an important role in regulating insect development. Here, we investigated the regulatory mechanism between the CncC/Keap1 pathway and metabolism of silkworm hormones in Lepidoptera. We found that high temperature induction accelerated the development of silkworm, the ecdysone content and related metabolic genes in hemolymph were significantly up-regulated, the CncC/Keap1 pathway was activated, and the expression of BmCncC was significantly increased, indicating that the Cncc/Keap1 pathway plays an important role in this process. The expression of cyp302a1 and cyp306a1 was significantly decreased by RNA interference with BmCncC, which indicated that CncC in silkworm had a regulatory relationship with downstream 20E synthetic gene. In summary, the results indicate that the CncC/Keap1 pathway plays an important role in regulating hormone metabolism in silkworm, providing a basis for further study of the relationship between CncC/Keap1 pathway and development in insects.

belongs to the Lepidoptera and is an important economic insect [7,8] which is susceptible to high 71 temperature environment during rearing, resulting in reduction of survival rate, cocoon rate, and 72 pupa yield [9,10]. The molecular mechanism of high temperature-mediated interaction between 73 metamorphosis processes and endogenous hormone metabolism has not been elucidated.

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The mammalian Nrf2 (nuclear factor erythroid 2 related factor 2)/Keap1 (Kelch-like 76 ECH-associated protein 1) pathway regulates intracellular redox potential, metabolic 77 detoxification, enhances cell resistance and delays aging [11,12]. Under stress conditions, Nrf2  The results indicate that high temperature treatment resulted in smaller body size of larvae and 98 transparent epidermis (Fig 1A), the characteristics and behavior of mature silkworm larvae were 99 observed either. In addition, the larvae maturity time was advanced 28±2 h in average and mostly 100 were concentrated around 144 h to 168 h (Fig 1C), indicating that high temperature treatment 101 shortens the developmental duration of larvae. Furthermore, the vitality and pupation rate in high 102 temperature group was reduced by 0.85 and 0.76-fold of the control group, respectively ( Fig 1B   103 and D), indicating that high temperature treatment reduced the vitality of the silkworm.

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The effect of HT stress on endogenous hormone and its metabolism-related genes expression

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The content of JH in hemolymph was reduced by 0.63-fold at 72 h after high temperature 108 treatment (Fig 2A), and the mRNA levels of the JH metabolism genes Met, JH3 and Kr-h1 were 109 down-regulated by 0.75, 0.77, and 0.54-fold, respectively ( Fig 2C). In addition, the content of 20E  temperature induction (Fig 1A), the survival and pupation rate of silkworm were significantly 147 decreased (Fig 1D), which was in consistent with previous studies [10]. BmCncC/keap1 pathway 148 plays an important role in response to oxidative stress and regulation of antioxidative and 149 detoxification genes expression [22]. Whether this pathway involved in metamorphosis processes 150 and regulates ecdysone-synthesis P450 genes in silkworm have not been reported. We found that 151 the duration of 5 th instar larva was shortened and the maturity time was advanced and relatively 152 concentrated after high temperature induction (Fig 1C) significantly down-regulated after high temperature treatment (Fig 2A and Fig 2C). The JH3 and Insects and treatment.

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The larvae of B. mori (Jingsong × Haoyue strain) maintained in our laboratory were reared on 207 mulberry leaves under 12 h light/ 12 h dark conditions at 25 ± 1°C with 75-85% humidity. After 208 three days of normal rearing, the 5 th instar silkworms were maintained at constant high 209 temperature of 36 °C and 75 ± 5% humidity, and feed with fresh mulberry leaves.

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Cell culture and treatment.

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The BmN cells were maintained at 27℃ in Grace insect medium, supplemented with 10% fetal

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Ecdysone and juvenile hormone (JH) content were determined using a kit (MEIMIAN,

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Shanghai, China), refer to the instructions. The absorbance value was measured at wavelength of 224 450 nm to calculate the sample content. All data are expressed as mean of 3 replicates.

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Total RNA was extracted from silkworm fat body using RNA lysate (Takara, China). Primer 228 sequences are shown in Table 1. Use Actin3 as the internal reference gene. QRT-PCR was 229 performed on a ViiA 7 System (ABI, Foster City, CA, USA). The reaction was in 20 μL volume.

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Amplification conditions were as follows: denaturation at 95 ℃ for 1 min, followed by 45 cycles 231 of 95℃ for 5 s, 55℃ for 10 s, and 72℃ for 10 s. Data are expressed as the mean of three 232 independent experiments ± SE (standard error).

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The samples of fat body of the control and treated groups were homogenized in lysis buffer