The circadian clock affects starvation resistance through the pentose phosphate pathway in silkworm, Bombyx mori.

Disruption of the circadian clock can affect starvation resistance, but the molecular mechanism is still unclear. Here, we found that starvation resistance was significantly reduced in the core gene BmPer deficient mutant silkworms (Per-/-), but the mutant's starvation resistance increased with larval age. Under natural physiological conditions, the weight of mutant 5th instar larvae was significantly increased compared to wild type, and the accumulation ability of triglycerides and glycogen in the fat bodies was upregulated. However, under starvation conditions, the weight consumption of mutant larvae was increased and cholesterol utilization was intensified. Transcriptome analysis showed that beta-oxidation was significantly upregulated under starvation conditions, fatty acid synthesis was inhibited, and the expression levels of genes related to mitochondrial function were significantly changed. Further investigations revealed that the redox balance, which is closely related to mitochondrial metabolism, was altered in the fat bodies, the antioxidant level was increased, and the pentose phosphate pathway, the source of reducing power in cells, was activated. Our findings suggest that one of the reasons for the increased energy burden observed in mutants is the need to maintain a more robust redox balance in metabolic tissues. This necessitates the diversion of more glucose into the pentose phosphate pathway to ensure an adequate supply of reducing power.

Analysis of 5-Year-old children's oral health service utilization and influencing factors in Guizhou Province, China (2019-2020).

BACKGROUND: This study aimed to investigate the utilization patterns and factors related to oral health care for 5-year-old preschoolers based on Andersen's Behavioural Model in Guizhou Province, Western China. METHOD: A cross-sectional study of 4,862 5-year-old preschoolers in 66 kindergartens was conducted in 2019 and 2020. A basic oral examination and a survey of parents and grandparents were conducted to gather data on oral health services. The results were analysed using chi-square tests and logistic regression analysis. RESULT: The utilization rate of oral health services for children in Guizhou province was 20.5%. The dmft was 4.43, and the rate of caries was 72.2%. The average cost of a dental visit was higher in rural areas and higher for girls. Logistic regression analysis revealed that dmft ≥ 6 teeth, a history of toothache, starting toothbrushing at age ≤ 3 years and limited parental knowledge were the primary factors impacting dental visits. CONCLUSION: Needs factors such as severe oral conditions and pain in children are the main reasons for the utilization of these services. This study underscores the urgency to actively promote the importance of oral health and expand insurance coverage for oral health services.

The clock gene Cryptochrome 1 is involved in the photoresponse of embryonic hatching behavior in Bombyx mori.

The hatching of insect eggs is a classic circadian behavior rhythm controlled by the biological clock. Its function is considered to impose a daily rhythm on the embryo, allowing it to hatch within a permissible time window. However, the molecular pathways through which the clock affects embryonic hatching behavior remain unclear. Here, we utilized a clock gene Cryptochrome1 (Cry1) knockout mutant to dissect the pathways by which the circadian clock affects embryonic hatching rhythm in the silkworm. In the Cry1 mutant, the embryo hatching rhythm was disrupted. Under the constant light or constant dark incubation conditions, mutant embryos lost their hatching rhythm, while wild-type embryos hatch exhibiting free-running rhythm. In the light-dark cycle (LD), the hatching rhythm of CRY1-deficient silkworms could not be entrained by the LD photoperiod during the incubation period. The messenger RNA levels and enzymatic activities of Cht and Hel in the mutant embryos were significantly reduced at circadian time 24 (CT24). Transcriptome analysis revealed significant differences in gene expression at CT24 between the Cry1 knockout mutant and the wild-type, with 2616 differentially expressed genes identified. The enriched Gene Ontology pathway includes enzyme activity, energy availability, and protein translation. Short neuropeptide F signaling was reduced in the CT24 embryonic brain of the mutant, the expression of the neuropeptide PTTH was also reduced and the rhythm was lost, which further affects ecdysteroid signaling. Our results suggested that the silkworm circadian clock affects neuropeptide-hormone signaling as well as physiological functions related to hatching, which may regulate the hatching rhythm.

Inhibition of Expression of the Circadian Clock Gene Cryptochrome 1 Causes Abnormal Glucometabolic and Cell Growth in Bombyx mori Cells.

Cryptochrome is the earliest discovered photoreceptor protein in organisms. However, the effect of CRY (BmCRY), the clock protein in Bombyx mori, on the body or cell metabolism remains unclear. In this study, we continuously interfered with the expression of the BmCry1 gene (Cry1-KD) in the silkworm ovary cell line (BmN), and the BmN cells developed abnormally, with accelerated cell growth and a smaller nucleus. Metabolomics was used to identify the cause of the abnormal development of Cry1-KD cells based on gas chromatography/liquid chromatography-mass spectrometry. A total of 56 differential metabolites including sugars, acids, amino acids, and nucleotides were identified in wild-type and Cry1-KD cells. KEGG enrichment analysis showed that BmCry1 knockdown resulted in significantly upregulated glycometabolism in BmN cells, indicated by glucose-6-phosphate, fructose-6-phosphate, and pyruvic acid levels. The activities of key enzymes BmHK, BmPFK, and BmPK as well as their mRNA levels further confirmed that the glycometabolism level of Cry1-KD cells was significantly increased. Our results show that a possible mechanism of BmCry1 knockdown leading to abnormal cell development is the elevated level of glucose metabolism in cells.

The LIM Domain Protein BmFHL2 Inhibits Egg Production in Female Silkworm, Bombyx mori.

The female Bombyx mori accumulates a large amount of egg proteins, mainly Vg and 30K, during egg formation to provide nutrition for embryo development. The synthesis and transport of Vg have been extensively studied, particularly the regulation of Vg transcription induced by 20E; however, the mechanism of 30K protein synthesis is poorly studied. As a model organism of the order Lepidoptera, B. mori has high reproduction potential. In the present study, we found that the FHL2 homologous gene (BmFhl2) in B. mori is involved in inhibiting female egg formation by influencing the synthesis of 30K protein. Interference of BmFhl2 expression in silkworm females increased 30K protein synthesis, accelerated ovarian development, and significantly increased the number of eggs produced and laid; however, the 20E pathway was inhibited. The transcription levels of Vg and 30Kc19 were significantly downregulated following BmFhl2 overexpression in the silkworm ovarian cell line BmN. The Co-IP assay showed that the potential binding protein of BmFHL2 included three types of 30K proteins (30Kc12, 30Kc19, and 30Kc21). These results indicate that BmFHL2 participates in egg formation by affecting 30K protein in female B. mori.

Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori.

The circadian clock plays an integral role in hormone biosynthesis and secretion. However, how the circadian clock precisely coordinates hormonal homeostasis to maintain normal animal development remains unclear. Here, we show that knocking out the core clock gene Cryptochrome 1 (Cry1) significantly delays the developmental time in Bombyx mori. This study focuses on the ecdysone and juvenile hormone signalling pathways of fifth instar larvae with the longest developmental time delay. We found that the mutant reduced prothoracicotropic hormone synthesis in the brain, and could not produce sufficient ecdysone in the prothoracic gland, resulting in a delayed peak of 20-hydroxyecdysone titre in the hemolymph of fifth instar larvae, prolonging developmental time. Moreover, further investigation revealed that the mutant enhanced juvenile hormone biosynthesis and signalling pathway and that this higher juvenile hormone titre also resulted in prolonged developmental time in fifth instar larvae. Our results provide insights into the molecular mechanisms by which the circadian clock regulates animal development by maintaining hormonal homeostasis.

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

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.

Dental Caries and Associated Factors in 3-5-Year-Old Children in Guizhou Province, China: An Epidemiological Survey (2015-2016).

Objective: This study aimed to explore the factors influencing dental caries among 3-5-year-old children in Guizhou Province and the interrelationship between these factors using structural equation modeling, while providing theoretical references to improve the prevention and control strategy. Materials and Methods: A total of 1,291 children aged 3-5 years in Guizhou Province were selected by a multistage stratified and whole group random sampling to examine the caries prevalence in whole-mouth deciduous teeth crowns, and parents were surveyed with questionnaires to analyze the caries-related factors. IBM SPSS Statistics v 23.0 software (IBM, Armonk, NY, USA) was used for statistical analysis. Results: The caries prevalence of children aged 3-5 years in Guizhou Province was 63.1%, the mean decayed-missing-filled teeth was 3.32, the caries filling rate was 0.5%, and there was no statistically significant difference between urban and rural areas and among genders in each age group; results of logistic regression analysis showed that the caries risk increased with the following factors: age, brushing frequency <2 times per day when parents did not take their children to the dentist, and with parents poor evaluation of the oral condition of their children. The higher the education of the parent, the lower the risk of children suffering from caries in deciduous teeth. Conclusions: With an overall poor situation about oral hygiene habits, oral healthcare attitude of the parents, and behavior transformation, the prevalence of dental caries in the deciduous teeth of children aged 3-5 years in Guizhou Province is high, and their caries status was severe, with more than 99% of the caries cases that were untreated. Therefore, prevention and treatment measures of caries in preschool children need strengthening through the improvement of public awareness and the enhancement of the management of oral health habits of their children.

Circadian Clock Gene Period Contributes to Diapause via GABAeric-Diapause Hormone Pathway in Bombyx mori.

Diapause is a developmental transition in insects based on seasonal adaptation to adversity; it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.

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

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.

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

Circadian clock system disorders can lead to uncontrolled cell proliferation, but the molecular mechanism remains unknown. We used a Bombyx mori animal model of single Period gene (BmPer) expression to investigate this mechanism. A slow growing developmental cell model (Per-KD) was isolated from a B. mori ovarian cell line (BmN) by continuous knock down of BmPer expression. The effects of BmPer expression knockdown (Per-KD) on cell proliferation and apoptosis were opposite to those of m/hPer1 and m/hPer2 in mammals. The knockdown of BmPer expression led to cell cycle deceleration with shrinking of the BmN cell nucleus, and significant inhibition of nuclear DNA synthesis and cell proliferation. It also promoted autophagy via the lysosomal pathway, and accelerated apoptosis via the caspase pathway.