Inhibition of Period Gene Expression Causes Repression of Cell Cycle Progression and Cell Growth in the Bombyx mori Cells

The circadian clock plays a critical role in the regulation of host immune defense. However, the mechanistic basis for this regulation is largely unknown. Herein, the core clock gene cryptochrome1 (cry1) knockout line in Bombyx mori, an invertebrate animal model, was constructed to obtain the silkworm with dysfunctional molecular clock, and the dynamic regulation of the circadian clock on the immune responsiveness within 24 h of Staphylococcus aureus infection was analyzed. We found that deletion of cry1 decreased viability of silkworms and significantly reduced resistance of larvae to S. aureus. Time series RNA-seq analysis identified thousands of rhythmically expressed genes, including immune response genes, in the larval immune tissue, fat bodies. Uninfected cry1 knockout silkworms exhibited expression patterns of rhythmically expressed genes similar to wild-type (WT) silkworms infected with S. aureus. However, cry1 knockout silkworms exhibited a seriously weakened response to S. aureus infection. The immune response peaked at 6 and 24 h after infection, during which "transcription storms" occurred, and the expression levels of the immune response genes, PGRP and antimicrobial peptides (AMPs), were significantly upregulated in WT. In contrast, cry1 knockout did not effectively activate Toll, Imd, or NF-κB signaling pathways during the immune adjustment period from 12 to 18 h after infection, resulting in failure to initiate the immune responsiveness peak at 24 h after infection. This may be related to inhibited silkworm fat body energy metabolism. These results demonstrated the dynamic regulation of circadian clock on silkworm immune response to bacterial infection and provided important insights into host antimicrobial defense mechanisms.

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“…Quantitative reverse transcription PCR (qRT-PCR) was performed as previously described (Qiu et al, 2019).…”

Section: Quantitative Reverse Transcription Pcrmentioning

confidence: 99%

Temporal transcriptome reveals that circadian clock is involved in the dynamic regulation of immune response to bacterial infection in Bombyx mori

et al. 2022

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“…As described in Qiu et al (2019), we extracted total RNA from the pupal fat body and removed DNA from the total RNA samples by using RNAisoTM Plus and Recombinant DNase I (Takara). Then we reverse transcribed RNA to complementary DNA with a PrimeScript™ RT reagent Kit (Takara) with a reaction volume of 20 μl (4 μl 5 × Prime Script Buffer, 1 μl 50 μM Oligo d (T) primer, 1 μl 100 μM random hexamers, 1 μl Prime Script RT Enzyme Mix I and 13 μl of RNase free water which contained 1 μg RNA template).…”

Section: Methodsmentioning

confidence: 99%

Bombyx mori can synthesize ascorbic acid through the l‐gulose pathway to varying degrees depending on developmental stage

Hou

Dai

Liang

et al. 2021

Arch Insect Biochem Physiol

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Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l‐ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l‐gulono‐1,4‐lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo‐like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h–E168 h in the late embryonic stage and P48 h–P144 h in the middle‐late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC‐free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h–E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo‐like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l‐gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

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“…After 8 h, the hemolymph was collected and diluted 5-10 times (v/v) with HbSS. Furthermore, 60 μL of diluted hemolymph was taken for hematocyte climbing test (Qiu et al, 2019); we stained the hemocytes using Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 594 dye (C10339, Invitrogen, Carlsbad, CA, USA). The cell nucleus was counter-stained with DAPI (C1006, Beyotime); red (590/615 nm) and blue fluorescence (364/454 nm) were observed microscopically.…”

Section: Blood Cell (Hemocyte) Stainingmentioning

confidence: 99%

“…We added 1,000 μL Fluo-3 AM (S1056, Beyotime) staining solution (5 μM), incubated for 30 min in the dark at 25°C and observed the green fluorescence (488/530 nm) after staining. Apoptosis was also detected by FCM (Qiu et al, 2019); hemocytes were collected according to the method described above for PI-FCM, and the percentage of apoptotic blood cells was detected by Annexin V, FITC Apoptosis Detection Kit (AD10, DOJINDO, Tokyo, Japan) and FC500.…”

Section: Detection Of Blood Cell Autophagymentioning

confidence: 99%

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Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model

Wang

et al. 2021

Preprint

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Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration (PPC). It is often clinically complicated by malignant diseases or severe infections. Research on the molecular mechanism of High PPC (HPPC) is scant. Here, an animal model of primary hyperproteinemia was constructed in an invertebrate, Bombyx mori, to investigate the effect of HPPC on circulating blood cells. We showed that HPPC affected blood cell homeostasis and enhanced blood cell phagocytosis, leading to increased reactive oxygen species levels, and induced programmed cell death that depended on the endoplasmic reticulum-calcium ion signaling pathway. HPPC induced the proliferation of blood cells, mainly granulocytes, by activating the JAK/STAT signaling pathway. Supplementation with endocrine hormone active substance 20E significantly reduced the impact of HPPC on blood cell homeostasis. Herein, we reported a novel signaling pathway by which HPPC affected blood cell homeostasis, which was different from hyperglycemia, hyperlipidemia, and hypercholesterolemia. In addition, we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early monitoring index for hyperproteinemia.

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Inhibition of Period Gene Expression Causes Repression of Cell Cycle Progression and Cell Growth in the Bombyx mori Cells

Cited by 7 publications

References 67 publications

Temporal transcriptome reveals that circadian clock is involved in the dynamic regulation of immune response to bacterial infection in Bombyx mori

Temporal transcriptome reveals that circadian clock is involved in the dynamic regulation of immune response to bacterial infection in Bombyx mori

Bombyx mori can synthesize ascorbic acid through the l‐gulose pathway to varying degrees depending on developmental stage

Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model

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