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.

Circadian factors likely influence the occurrence, development, therapy, and prognosis of cardiovascular diseases (CVDs). To determine the association between the heart rate (HR) diurnal parameters and CVD risks, we designed an analytical strategy to detect diurnal rhythms of HR using longitudinal data collected by clinically used Holter monitors and wearable devices. By combining in-house developed algorithms with existing analytical tools, we obtained trough phase and nocturnal variation in HR for different purposes. The analytical strategy is robust and also sensitive enough to identify variations in HR rhythms influenced by multiple effectors such as jet lag, geological location and altitude, and age from total 211 volunteers. A total of 10,094 sets of 24-h Holter ECG data were analyzed by stepwise partial correlation to determine the critical points of HR trough phase and nocturnal variation. The following HR diurnal patterns correlate with high CVD risk: arrhythmic pattern, anti-phase pattern, rhythmic patterns with trough phase less than 0 (extremely advanced diurnal pattern) or more than 5 (extremely delayed diurnal pattern), and nocturnal variation less than 2.75 (extremely low) or more than 26 (extremely high). In addition, HR trough phases from wearable devices were nearly identical to those from 24-h Holter monitoring from 12 volunteers by linear correlation and Bland-Altman analysis. Our analytical system provides useful information to identify functional diurnal patterns and parameters by monitoring personalized, HR-based diurnal changes. These findings have important implications for understanding how a regular heart diurnal pattern benefits cardiac function and raising the possibility of non-pharmacological intervention against circadian related CVDs. With the rapid expansion of wearable devices, public cardiovascular health can be promoted if the analytical strategy is widely applied.

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.

The habitat of Chinese mitten crabs (CMCs, Eriocheir sinensis) has significant impact on their flavor and market price, and the characteristic flavor of CMCs produced in Yangcheng Lake in the lower reach of the Yangtze River in China is the most sought after by consumers. In this study, the differences in the intestinal microbiome and flavor amino acids, nucleotides, and organic acids in CMCs cultivated in Yangcheng Lake and Gucheng Lake (also in the lower reach of the Yangtze River) using the same model were compared. The results showed that CMCs cultivated in different lakes contained specific microbiota, with significantly different composition and abundance of the dominant microbiota. Although remarkable differences were noted in the contents of flavor amino acids, nucleotides, and lactic acid among different tissues and genders of CMCs, only some of these indicators varied between CMCs of different habitats, with respect to the distinctive flavor of CMCs. Thus, the difference in the intestinal microbiota of CMCs from different habitats may be related to the variation in the distinctive flavor of CMCs.

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.

School of Biology and Basic Medical Scienc Suzhou 215123, China. E-mail: szsqxu@sud Institute of Agricultural Biotechnology & Ec 215123, China College of Forestry, Shandong Agricultura China. E-mail: wzcui88@163.com National Engineering Laboratory for Mod Suzhou 215123, China † Electronic supplementary informa 10.1039/c8ra04627a ‡ These authors contributed equally to th Cite this: RSC Adv., 2018, 8, 26188

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.

Hatching behavior is a key target in silkworm (Bombyx mori) rearing, especially for the control of Lepidoptera pests. According to previous research, hatching rhythms appear to be controlled by a clock mechanism that restricts or "gates" hatching to a particular time. However, the underlying mechanism remains elusive. Under 12-h light:12-h dark photoperiod (LD) conditions, the transcriptional levels of the chitinase5 (Cht5) and hatching enzyme-like (Hel) genes, as well as the enzymatic activities of their gene products, oscillated in time with ambient light cycles, as did the transcriptional levels of the cryptochrome 1, cryptochrome 2, period (per), and timeless genes, which are key components of the negative feedback loop of the circadian rhythm. These changes were related to the expression profile of the ecdysteroid receptor gene and the hatching behavior of B. mori eggs. However, under continuous light or dark conditions, the hatching behavior, the expression levels of Cht5 and Hel, as well as the enzymatic activities of their gene products, were not synchronized unlike under LD conditions. In addition, immunohistochemistry experiments showed that light promoted the translocation of PER from the cytoplasm to the nucleus. In conclusion, LD cycles regulate the hatching rhythm of B. mori via negative feedback loop of the circadian oscillator.

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.

The lactic acid and ROS content in tissues of Eriocheir sinensis raised in lake and pond were investigated in this paper.The results showed that, the lactic acid content in gonad, liver and muscle from Lake Crabs, no matter Yangcheng Lake or Taihu Lake, were extremely lower than that from pond around the lakes, while the lactic acid content of the top grade mature crabs were lower than that in small crabs.At the same time, lactic acid content in male were lower than that in female, and the lactic acid existed more in muscles than in the other two kinds of tissue.Moreover, the lactic acid in tissues fell earlier and more rapidly in lake crabs than that in pond crabs from September to November.The ROS detection in muscle reveals that the top grade mature crabs contain less ROS than small female crabs, and the male one al-ways had less ROS than female one of the same size.The result elucidated that lactic acid, ROS content or the indicator of oxidation resistance could be a matter of concern in the quality and taste of Yangcheng Lake crab, and they deserved atlen-tion and in-depth study.

The physiology and metabolism of poikilothermic insects are under the control of environmental temperature. Temperature is the primary cue for the circadian rhythm. Reports on the timing mechanisms of temperature in lepidopterans are limited. This study used Bombyx mori BmN ovarian cells to investigate the effect of temperature on expression of the main circadian clock genes in a negative feedback loop. A 37°C, 30-min high-temperature stimulation induced transcription of the circadian clock genes Cry1, Cry2, Per, and Tim. The gene expression profiles showed rhythmic oscillations, with shortened oscillatory periods for Cry1 and altered oscillatory phases for Cry1 and Per. Cyclical increases in temperature of 2°C starting at 26°C, 5°C starting at 20 or 25°C, or 10°C starting at 20°C induced cyclical changes in expression and protein from the four circadian clock genes. Cyclical temperature changes with a difference of 10°C had the most influence. In conclusion, cyclical changes in temperature with differences from 2 to 10°C reset and synchronized the circadian clock of silkworm BmN cells. Transcription of the genes for and protein from Cry2 and Per showed a better reset and synchronization with cyclical temperature changes.

The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval–pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval–pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals.

The silk gland of Bombyx mori (BmSG) has gained significant attention by dint of superior synthesis and secretion of proteins. However, the application of BmSG bioreactor is still a controversial issue because of low yields of recombinant proteins. Here, a 3057 bp full-length coding sequence of Hpl was designed and transformed into the silkworm genome, and then the mutant (Hpl/Hpl) with specific expression of Hpl in posterior BmSG (BmPSG) was obtained. In the mutants, the transcription level of Fib-L and P25, and corresponding encoding proteins, did not decrease. However, the mRNA level of Fib-H was reduced by 71.1%, and Fib-H protein in the secreted fibroin was decreased from 91.86% to 71.01%. The mRNA level of Hpl was 0.73% and 0.74% of Fib-H and Fib-L, respectively, while HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted silk protein. The exogenous protein was therefore very efficiently translated and secreted. Further analysis of differentially expressed gene (DEG) was carried out in the BmPSG cells and 891 DEGs were detected, of which 208 genes were related to protein metabolism. Reduced expression of endogenous silk proteins in the BmPSG could effectively improve the production efficiency of recombinant exogenous proteins.

以阻止熟蚕吐丝的方法建立家蚕高蛋白血症模型,研究模型家蚕丝腺退化过程中细胞程序性死亡的特点,探索家蚕高蛋白血症模型的发病机制.形态解剖学观察显示,模型家蚕的丝腺退化过程延长了2倍时间;组织切片HE染色显示,模型家蚕的中部丝腺和后部丝腺都出现了退化启动推迟的现象.对家蚕组织解体退化信号通路相关基因表达水平的检测显示,模型家蚕丝腺细胞中自噬信号基因Atg6和Atg8的转录水平显著低于对照组家蚕,而凋亡信号基因Dronc的转录水平则显著高于对照组家蚕.免疫组化染色显示,模型家蚕丝腺细胞中凋亡启动蛋白Caspase-3在丝腺退化过程中持续表达.研究结果表明高蛋白血症模型家蚕丝腺细胞的凋亡活动增强,而丝腺退化延缓与自噬作用启动延迟有关.

鳞翅目昆虫生物钟环路的核心成员,在蛋白质分子进化和相互作用网络等方面,与哺乳类,甚至与果蝇等经典模式昆虫有较大差异.总结了昼夜节律对鳞翅目昆虫孵化与进食、生长与变态、生殖与滞育、呼叫与迁徙等生理行为的影响,以及核心钟基因Cry、Per和Tim等的研究进展,分析了鳞翅目昆虫生物钟负反馈环路与哺乳动物和果蝇的差异,介绍了鳞翅目昆虫作为温度授时的生物钟负反馈环路研究和外周生物钟分子机制研究等方面的材料优势.

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third-instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20-hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry-leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin-synthesis-dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.

Cryptochrome gene(Cry) is one of the major biological clock genes which were widely distributed in bacteria and eukaryotes.Cry genes of insect species are clearly divided into two types,Cry1 and Cry2.Only Cry1 is expressed in Drosophila,while only Cry1 was expressed in bees and other hymenopteran insects.In order to explore the molecular mechanism of circadian clock in lepidopteran model insect Bombyx mori and the evolution of CRY proteins in insect species,we cloned the cDNA sequences of Bmcry1(2 166 bp,GenBank accession no.HM747059) and Bmcry2(2 389 bp,GenBank accession no. HM747060),and obtained their gene sequences(GenBank accession no.HM747057 and HM747058, respectively) by sequence alignment and assembly.Bmcry1 have 12 exons and 11 introns,while Bmcry2 have 9 exons and 8 introns.Chromosome mapping showed that Bmcry1 and Bmcry2 were located on chromosome 17 and chromosome 15,respectively.We predicted the three-dimensional structure of Bmcry1 and Bmcry2 by homology modeling.The results showed that the FAD entrances are large and deep, consistent with the fact that CRY proteins do not bind with pyrimidine dimers,and the surfaces of Bmcry1 and Bmcry2 are more negatively charged,only FAD entrances have accumulated positive charge.Moreover, we researched the molecular evolution of Bmcry1 and Bmcry2 by multiple sequence alignment,protein motif analysis,functional domain analysis and cluster analysis.The results showed that Bmcry1 and Bmcry2 belonged to insect CRY1 and CRY2,respectively,and were closely to the corresponding proteins in other lepidopteran insects like Antheraea pernyi.Similar to the CRY proteins in other insects,Bmcry1 and Bmcry2 have DNA photolysis enzyme domain and FAD binding domain.However,the two domains have different conservative sites between CRY1 and CRY2 in insect species,and their protein motifs are also different.Our experiment provided a basis for further investigating the mechanism of molecular evolution and function of CRY1 and CRY2 in B.mori.

Bombyx mori is the model insect of lepioloptera with diapausing during its egg stage.Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs.to investigate the relationship of diapause inducements and temperature and photoperiod,we design this experiment to search the affect of temperature and photoperiod on the gene sod and cat.When eggs were incubated in 25 LL,their sod mRNA would keep at a high level,but the transcription level was rapidly down-regulated at EAT1 800℃.h and 3 600℃.h.The expression of sod gene in 20 LD was significantly higher than in 15 DD,this phenomenon was more obvious when the embryonic nervous had fully grown in the later incubation stage.The expression of cat gene had no obvious differences when eggs were incubated in 25 LL,20 LD or 15 DD but it was notable that the mRNA level of cat all increased in the late incubation stage,which was in accordance with the fact that the eggs were more sensitive to diapause-inducing incubation temperature and photoperiod of this time.After artificial hatching treatment with hydrochloric acid at 24h after oviposition,the sod mRNA in diapause-terminated eggs (DTE) was much higher than diapause-destined eggs (DDE).From 24 to 192hours after oviposition,eggs at progeny went into the diapause stage,the transcription level of sod subsequently decreased.After we examined the common effect of illumination and temperature on the sod gene of progeny eggs,it was observed that the sod mRNA increased under constant darkness at 15℃.The cat mRNA level of DTE was lower than DDE at first after oviposition,but then the mRNA increased,which indicated that the expression of cat might be inhibited by hydrochloric acid at the early activation.Then we investigated the common effect of illumination and temperature on the cat gene of progeny eggs and found that the expression level of cat in 20 LD was lower than in 25 LL and 15 DD.

Diapause hormone (DH) is the key factor that leads to diapause in silkworm (Bombyx mori).In present study,expressional variations of silkworm diapause hormone gene (Dh) caused by various temperature and photoperiod conditions were examined to explore the regulatory mechanism of temperature and illumination to silkworm diapause.The results showed that Dh was steadily and highly expressed in developmental embryos under constant illumination at 25 ℃ (25LL),but lowly expressed under constant darkness at 15 ℃ (15DD).Under 12 h illumination and 12 h darkness cycle at 20 ℃ (20LD) or 25LL,the Dh mRNA level of embryos was up-regulated significantly at the late developmental stage (after the effective accumulated temperature was beyond 2 160 ℃·h),being consistent with the super sensitivity of embryos to external illumination and temperature at this stage.Dh was dramatically up-regulated at 25 ℃ during the postembryonic stage,and illumination could also up-regulate the Dh expression level during pupal and moth stages.And the regulation by high temperature was stronger than that by illumination.In addition,gene microarray analysis showed that Dh had sex-and tissue-specific expression characteristics on day 3 of the 5th instar larvae.The Dh gene's mRNA transcriptional levels in multiple tissues of female larvae were significantly lower than those of the male larvae,and were very low in ovary.The incubation temperature and photoperiod of parental eggs had influence on the diapause of offspring eggs,and also affected the mRNA level of Dh.However,the mRNA transcriptional level of Dh gene in offspring eggs was not the key factor to determine whether diapause of the eggs occurred.It was suggested that the sex difference of silkworm Dh gene expression and the discrepancy between the expression pattern of Dh gene in silkworm eggs and the diapause of the eggs were relaxed to splicing of precursor polypeptide of the Dh gene.