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.

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.

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.

Differences in gut microbiota between silkworms (Bombyx mori) reared on fresh mulberry (Morus alba var. multicaulis) leaves or an artificial diet.

Artificial diets for silkworms have many potential applications and they are important in sericulture. However, the challenges of weak larvae and low silk protein synthesis efficiency in silkworms reared on artificial diets have not been resolved. Here, we used high-throughput sequencing to analyse the differences between the gut microbiota of 5th-instar larvae reared on mulberry leaves and larvae reared on an artificial diet. The results showed that at the phylum level, Cyanobacteria, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria are the dominant bacteria in the intestines of silkworm larvae of all the strains. But the abundance of dominant bacteria in the gut microbiota differed between the silkworm strains that were reared on mulberry leaves, as well as between the silkworm strains that were reared on the artificial diet, while the gut microbiota diversity was lower in the silkworm strains that were reared on the artificial diet. Prediction of the functions of the gut microbiota in the hosts indicated that there was no significant difference between the silkworm strains that were reared on mulberry leaves, while there were significant differences between silkworm strains reared on the artificial diet. When the silkworm diet changed from mulberry leaves to the artificial diet, changes in gut microbiota in the silkworms affected host nutrient metabolism and immune resistance. These changes may be related to the adaptation of silkworms to their long evolutionary history of eating mulberry leaves.

Metabolomics differences between silkworms (Bombyx mori) reared on fresh mulberry (Morus) leaves or artificial diets.

Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography-mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

Inhibition of expression of the circadian clock gene Period causes metabolic abnormalities including repression of glycometabolism in Bombyx mori cells.

Abnormalities in the circadian clock system are known to affect the body's metabolic functions, though the molecular mechanisms responsible remain uncertain. In this study, we achieved continuous knockdown of B. mori Period (BmPer) gene expression in the B. mori ovary cell line (BmN), and generated a Per-KD B. mori model with developmental disorders including small individual cells and slow growth. We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism. Amino acids that used glucose metabolites as a source were also down-regulated, while lipid metabolism and nucleotide metabolism were significantly up-regulated. Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism, as well as to metabolites such as aspartate, alanine, and xanthine in other pathways. Further validation experiments showed that the activities of the key enzymes of glucose metabolism, hexokinase, phosphofructokinase, and citrate synthase, were significantly decreased and transcription of their encoding genes, as well as that of pyruvate kinase, were also significantly down-regulated. We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism, and may be associated with changes in cellular amino acid metabolism, and in cell growth and development.