Fat Body Of Silkworm Research Articles

BmEL-2 promotes triglyceride metabolism by regulating BmAGPATγ and BmFAF2 expression in Bombyx mori.

Bombyx mori ELAV-like-1 (BmEL-1) and B. mori ELAV-like-2 (BmEL-2) are 2 members of the ELAV-like family of RNA-binding proteins. Mutations in Bmel-1 and Bmel-2 resulted in 5.8% and 28.5% decreases in larval weight on the 3rd day of the 5th instar larva (L5D3), respectively. Triglycerides (TG) are the most important energy resource and are the main component of neutral fat (NF) in animals. To investigate the role of Bmelav-like genes in the synthesis and decomposition of TG, transcriptomic, and metabolic analyses were performed on the whole bodies on the 1st day of the 2nd instar larvae (L2D1) and on fat bodies on L5D3 of Bmel-1- and Bmel-2- mutants, respectively. As compared with the control silkworm, differentially expressed genes generated in both mutants were mainly enriched in lysine degradation, fatty acid (FA) metabolism, and unsaturated FAs biosynthesis. The diglyceride and phosphatide contents were significantly lower in Bmel-1- and Bmel-2- fat bodies than those of the control group. Consistently, the NF content of both mutants' fat bodies were reduced by 50% and 60%, respectively. BmEL-2 positively regulates BmAGPATγ (B. mori 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma, LOC101741736) and BmFaF2 (B. mori fatty acid synthetase-associated factor 2, LOC101739090) expression by binding to the specific regions of their 3' untranslated regions in BmN cells. This study suggests that BmEL-2 may be an important regulator of BmAGPATγ and BmFAF2 expression and thereby participates in TG metabolism in the silkworm fat body.

Poria cocos polysaccharides alleviate cyclophosphamide-induced immunosuppression effects in silkworm and their metabolic profiling analysis

The present study investigated the immune regulatory mechanisms of Poria cocos polysaccharides (PS) in a cyclophosphamide (CTX)-induced immunosuppression silkworm model. We found that PS increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, as well as reactive oxygen species (ROS) levels in the fat body or haemocytes of CTX-treated silkworms. PS also strongly stimulated the immunity of silkworms by increasing haemocyte counts and PO activity and upregulating the mRNA expression of immune-related genes. We further investigated the metabolic profile of the fat body after PS treatment using liquid chromatography–massspectrometry/mass spectrometry (LC–MS/MS) and identified 236 differentially expressed metabolites. Among them, nine metabolites related to amino acid metabolism were present at high levels in the PS-treated group, whereas the contents of metabolites involved in sphingolipid metabolism and alpha-linolenic acid metabolism were significantly decreased after PS treatment. The amino acid metabolism pathways and lipid metabolism pathways were found to be significantly altered in the PS-treated group, which indicates that PS may regulate the immune system of silkworms by reprogramming lipid and amino acid metabolism. This study provides new insight into the immune regulatory effect of PS in animals.

Domestication Gene Mlx and Its Partner Mondo Are Involved in Controlling the Larval Body Size and Cocoon Shell Weight of Bombyx mori.

Bombyx mori was domesticated from Bombyx mandarina. The long-term domestication of the silkworm has brought about many remarkable changes to its body size and cocoon shell weight. However, the molecular mechanism underlying the improvement in the economic characteristics of this species during domestication remains unclear. In this study, we found that a transposable element (TE)-Bm1-was present in the upstream regulatory region of the Mlx (Max-like protein X) gene in wild silkworms but not in all domesticated silkworms. The absence of Bm1 caused an increase in the promoter activity and mRNA content of Mlx. Mlx and its partner Mondo belong to the bHLHZ transcription factors family and regulate nutrient metabolism. RNAi of Mlx and Mondo decreased the expression and promoter activity of glucose metabolism-related genes (trehalose transport (Tret), phosphofructokinase (PFK), and pyruvate kinase (PK)), lipogenic genes (Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)), and glutamine synthesis gene (Glutamine synthase 2, (GS2)). Furthermore, the transgenic overexpression of Mlx and Mondo in the fat body of silkworms increased the larval body size, cocoon shell weight, and egg number, but the silencing of the two genes resulted in the opposite phenotypes. Our results reveal the molecular mechanism of Mlx selection during domestication and its successful use in the molecular breeding of Bombyx mori.

Cloning of suppressor of cytokine signaling 7 from silkworm (Bombyx mori) and its response to the infection of Bombyx mori nucleopolyhedrovirus.

Suppressors of cytokine signaling (SOCS) play important roles in the regulation of growth, development, and immunity of eukaryotic organisms. SOCS7 is an important member of the SOCS family, but its physiological and pathological functions remain largely unknown in invertebrates including insects. Here, we first report the cloning of a SOCS7 gene from a domesticated silkworm (Bombyx mori), named BmSOCS7. We have characterized BmSOCS7 expression profiles in silkworm varieties susceptible or resistant to the infection of Bombyx mori nucleopolyhedrovirus (BmNPV) using the real-time fluorescence quantitative PCR. BmSOCS7 expresses highly in embryogenesis and lowly in metamorphosis in resistant silkworms but does in opposite contrast in susceptible silkworms. Its expression is at very low level in the fat body of resistant silkworms but is relatively high in the fat body of susceptible ones. BmNPV inoculation induces a transient downregulation and then a general upregulation of BmSOCS7 expression in BmN cells, while it induces a general downregulation in silkworm midgut, fat body and hemolymph with more pronounced effect in resistant silkworms than susceptible ones and more prominent in the fat body and hemolymph than the midgut. Together, our work reveals that downregulation of BmSOCS7 expression may be an important strategy for silkworm anti-BmNPV immune response, and BmSOCS7 may mainly function in the fat body and hemolymph rather than the midgut to participate in BmNPV infection process.

Characterization of a glutamate-cysteine ligase in Bombyx mori.

Glutamate-cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase, and its essential role in detoxification and resistance to oxidative stress, GSH synthesis has not been described in Bombyx mori (silkworms) to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis by GCL in silkworms, which may help in developing insecticides to tackle agricultural pests. Based on the amino acid sequence and phylogenetic tree, the B. mori GCL belongs to group 2, and is designated bmGCL. Recombinant bmGCL was overexpressed and purified to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg2+ to ligate glutamate and cysteine. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B irradiation. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. In this study, we characterized the B. mori GCL and analyzed its biochemical properties. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in the silkworms.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

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 24h 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 24h 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 18h after infection, resulting in failure to initiate the immune responsiveness peak at 24h 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.

Cellular Lnc_209997 suppresses Bombyx mori nucleopolyhedrovirus replication by targeting miR-275-5p in B. mori.

Long non-coding RNA (lncRNA) is a type of non-coding RNA molecule, which exceeds 200 nucleotides in length and participates in the regulation of a variety of life activities. Recent studies showed that lncRNAs play important roles in viral infection and host immunity. At present, the researches on insect lncRNAs are relatively few. In this study, we found the expression of Lnc_209997 was significantly down-regulated in silkworm fat body infected with Bombyx mori nucleopolyhedrosis virus (BmNPV). Inhibition of Lnc_209997 promoted BmNPV replication. Enhancing the expression of Lnc_209997 inhibited the proliferation of BmNPV. miR-275-5p was up-regulated in silkworm fat body infected with BmNPV. Dual luciferase reporter gene system confirmed the interaction between Lnc_209997 and miR-275-5p. Over-expression of Lnc_209997 inhibited the expression of miR-275-5p, while inhibition of Lnc_209997 enhanced the expression of miR-275-5p. Further, over-expression of miR-275-5p can facilitate the replication of BmNPV. These results suggested that BmNPV could increase the expression of miR-275-5p by inhibiting cellular Lnc_209997 expression to promote their own proliferation. Our results are helpful for better understanding the role of lncRNAs in BmNPV infection, and provide insights into elucidating the molecular mechanism of interaction between Bombyx mori and virus.

Transcriptome Analysis Reveals the Gene Expression Changes in the Silkworm (Bombyx mori) in Response to Hydrogen Sulfide Exposure.

Simple SummaryThe fat body is one of the most important tissues in the body of insects due to its number of functions. Nowadays the new physiological function of H2S has gained attention as a novel signaling molecule. H2S performs crucial regulatory functions involving growth, the cardiovascular system, oxidative stress, and inflammation in many organisms. In this study, RNA-seq technology was used to investigate the fat body of the silkworm at the transcriptional level after H2S exposure during the 5th larvae stage. A total of 1200 (DEGs) was identified after 7.5 µM H2S treatment, of which 977 DEGs were up-regulated and 223 DEGs were down-regulated. DEGs were mainly involved in the transport pathway, cellular community, carbohydrate metabolism, and immune-associated signal transduction. Present research provides new insights on the gene expression changes in the fat body of silkworms after H2S exposure.Hydrogen sulfide (H2S) has been recognized for its beneficial influence on physiological alterations. The development (body weight) and economic characteristics (cocoon weight, cocoon shell ratio, and cocoon shell weight) of silkworms were increased after continuous 7.5 µM H2S treatment. In the present study, gene expression changes in the fat body of silkworms at the 5th instar larvae in response to the H2S were investigated through comparative transcriptome analysis. Moreover, the expression pattern of significant differentially expressed genes (DEGs) at the 5th instar larvae was confirmed by quantitative real-time PCR (qRT-PCR) after H2S exposure. A total of 1200 (DEGs) was identified, of which 977 DEGs were up-regulated and 223 DEGs were down-regulated. Most of the DEGs were involved in the transport pathway, cellular community, carbohydrate metabolism, and immune-associated signal transduction. The up regulated genes under H2S exposure were involved in endocytosis, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), and the synthesis of fibroin, while genes related to inflammation were down-regulated, indicating that H2S could promote energy metabolism, the transport pathway, silk synthesis, and inhibit inflammation in the silkworm. In addition, the expression levels of these genes were increased or decreased in a time-dependent manner during the 5th instar larvae. These results provided insight into the effects of H2S on silkworms at the transcriptional level and a substantial foundation for understanding H2S function.

Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori)

Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 μg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.

Protective effects of pretreatment with Fe2+, Cu2+, and Rb+ on phoxim poisoning in silkworm, Bombyx mori

BackgroundPhoxim is a widely used organophosphorus pesticide in agriculture. People are paying more and more attention to its toxicity. At present, there is no appropriate way to solve the phoxim poisoning of silkworm, which severely affected the development of sericulture. Fe2+, Cu2+, Rb+ exerted their biological effects through various forms in vivo. MethodsTo evaluate the effect of Fe2+/Cu2+/Rb+ on phoxim poisoning in silkworm, Bombyx mori were treated with fresh mulberry leaves soaked in 2.5 mg/L phoxim for 2 min with 50 mg/L FeCl2, 150 mg/L CuCl2, or 0.5 mg/L RbCl from 5 days of the fifth-instar silkworm. ResultsFe2+, Cu2+, and Rb+ pretreatments significantly inhibited the phoxim-induced reduction of survival rate and alleviated the phoxim-induced poisoning symptoms. The protective effects of Fe2+, Cu2+, and Rb+ on phoxim poisoning might be due to their enhancement of superoxide dismutase (SOD), catalase (CAT), and carboxylesterase (CarE) in the hemolymph and fat body of silkworm. This enhancement might reduce reactive oxygen species (ROS) accumulation and oxidative stress (OS) caused by phoxim poisoning. Thereby it reduced the damage to silkworm tissues and cells. ConclusionsThese results showed that Fe2+, Cu2+, and Rb+ treatments protected the silkworm from phoxim poisoning by directly enhancing the activity of SOD, CAT, and CarE enzymes and reducing oxidative stress, but not dependent on the high expression of CYP genes. The use of Fe2+, Cu2+, and Rb+ to enhance the activity of SOD, CAT, and CarE enzymes may be an underlying effective way to solve phoxim poisoning in the silkworm industry.

Evaluation of Antibacterial Drugs Using Silkworms Infected by Cutibacterium acnes.

Simple SummaryCutibacterium acnes, a common bacterium on human skin, is a causative agent of inflammatory skin diseases and systemic infections. Systemic infections caused by C. acnes are difficult to treat because of the drug resistance to typical macrolides. The development of a systemic infection model for C. acnes contributes to searching candidates for drug discovery. Silkworm is a useful model animal for evaluating the efficacy of compounds. Several silkworm infection models have been established for the identification of virulence-related genes and novel antibacterial drugs. However, the systemic infection model of C. acnes using silkworms was not yet established. We established a new silkworm infection model with C. acnes and evaluated the efficacy of antibacterial drugs using the silkworm infection model. The silkworm infection model might be used to identify drug candidates for the treatment against C. acnes infection.Cutibacterium acnes is a causative agent of inflammatory skin diseases and systemic infections. Systemic infections caused by C. acnes are difficult to treat, and the development of a systemic infection model for C. acnes would be useful for elucidating the mechanisms of infection and searching for therapeutic agents. In this study, we established a silkworm infection model as a new experimental system to evaluate the interaction between C. acnes and the host, and the efficacy of antibacterial drugs. Silkworms infected with C. acnes died when reared at 37 °C. The dose of injected bacterial cells required to kill half of the silkworms (LD50) was determined under rearing conditions at 37 °C. The viable cell number of C. acnes was increased in the hemolymph and fat body of the infected silkworms. Silkworms injected with autoclaved C. acnes cells did not die during the study period. The survival time of silkworms injected with C. acnes was prolonged by the injection of antibacterial drugs such as tetracycline and clindamycin. These findings suggest that the silkworm C. acnes infection model can be used to evaluate host toxicity caused by C. acnes and the in vivo efficacy of antimicrobial drugs.

Genomics and proteomics combined analysis revealed the toxicity response of silkworm Bombyx mori to the environmental pathogen Bacillus cereus ZJ-4

Bacterial contamination has caused a major public health problem worldwide. Bacillus cereus is a conditional environmental pathogenic bacteria that can cause food poisoning. Whether environmental pathogens can cause widespread transmission in the insect kingdom is unclear. In this study, a Bacillus cereus ZJ-4 was isolated from the hospital environment of Zhenjiang City, Jiangsu Province, China. It was fatal by injection into the silkworm hemolymph. To investigated the potential toxic factors of ZJ-4 and clarified the toxicity response mechanism of silkworm by the ZJ-4 infection. Then, the whole genome of ZJ-4 was sequenced, and the immune mechanism of silkworm fat body to ZJ-4 pathogen was studied by HE pathological section and proteomics. Bacterial genome sequencing indicated that ZJ-4 had 352 drug resistance genes and 6 virulence genes. After 36 h of subcutaneous puncture with ZJ-4 suspension, the pathological changes were obviously found in HE pathological sections of fat body tissue. Comparative proteomic results indicated that differentially expressed proteins are mainly involved in stress reactions, biological regulation, and innate immunity. The qRT-PCR analysis showed that the expressions of β-GRP, Spaetzle, MyD88, Tube and Dorsal genes in Toll pathway were up-regulated, while Pell and Cactus genes were down-regulated; in the antimicrobial peptide pathway, Glv2, Lzm, Mor, and Leb3 genes were up-regulated, while attacin1 and defensin genes were down-regulated; Sod gene was up-regulated, while Cat gene was down-regulated in the antioxidant pathway; Ldh, Sdh, and Mdh genes were down-regulated in glucose metabolism pathway. These results indicated that ZJ-4 can damage the innate immune pathway of silkworm, and also affect the normal immune function of fat body cells.

Modulating the Metabolic Activity of Silkworm, Bombyx mori L. with the Supplementation of Honey and Protein Sources

The present study aimed to assess the effect of honey obtained from different bee species and protein sources on the metabolic changes in mulberry silkworm, Bombyx mori L. The results of experiment showed that oral administration of honey and protein sources to silkworm larvae during late-age instars daily once significantly changed metabolic processes. Among the various treatments and concentrations tested, 5 per cent Trigona iridipennis honey significantly increased the carbohydrate in haemolymph (16.71 mg/mL) and fat body (12.38 mg/g) over all other treatments. The lipid content in haemolymph (23.03 mg/mL) and fat body (45.38 mg/g) of silkworm were also found to be superior in the larval batch treated with 5 per cent T. iridipennis. Apart from this, the same treatment was showed statistical superiority in increasing the activity of enzymes viz., aspartate aminotransferase (269.78 µg/mL) and alanine aminotransferase (563.25 µg/mL), which play an important role in protein metabolism.

Ni-modified magnetic nanoparticles for affinity purification of His-tagged proteins from the complex matrix of the silkworm fat body

Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.

The piRNA response to BmNPV infection in the silkworm fat body and midgut.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that causes huge losses to the silkworm industry but the piRNA responses during BmNPV infection in the silkworm remain uninvestigated. Here, silkworm piRNA profiles of uninfected and BmNPV-infected fat body and midgut were determined by high-through sequencing in the early stages of BmNPV infection. A total of 2675 and 3396 genome-derived piRNAs were identified from fat body and midgut, respectively. These genome-derived piRNAs mainly originated from unannotated instead of transposon regions in the silkworm genome. In total, 572 piRNAs were associated with 280 putative target genes in fat body and 805 piRNAs with 380 target genes in midgut. Compared to uninfected tissues, 322 and 129 piRNAs were significantly upregulated in BmNPV-infected fat body and midgut, respectively. In addition, 276 and 117 piRNAs were significantly downregulated. Moreover, differentially expressed (DE) piRNAs during BmNPV infection differed significantly between fat body and midgut. Putative DE piRNA-targeted genes were associated with "response to stimulus" and "environmental information processing" in fat body after infection with BmNPV, which may indicate an active piRNA response to BmNPV infection in fat body. This study may lay the foundation for future research of the potential roles of the piRNA pathway and specific piRNAs in BmNPV pathogenesis.

Effects of pyriproxyfen exposure on immune signaling pathway and transcription of detoxification enzyme genes in fat body of silkworm, Bombyx mori

Sericulture is a very important and flourishing industry in developing countries. Bombyx mori is a kind of important and well-studied economic insects in the whole world. In China, applying of pyriproxyfen pesticide often resulted in non-cocooning and silk yield reduction. However, the effects of pyriproxyfen exposure on immune signaling pathway in fat body of silkworm has not been reported yet now. In the present study, we found that the growth and development of silkworm were significantly affected by pyriproxyfen exposure and the fat body tissues were injured after treatment. It was also showed that the expressions of key genes of PI3K/Akt and CncC/Keap1 pathway can be elevated at 24–96 h after pyriproxyfen exposure. Furtherly, the relative expression levels of detoxification enzyme genes and the activities of detoxification enzymes were both increased by pyriproxyfen exposure. These results provided comprehensive view of fat body injury and gene expression changes in silkworm after pyriproxyfen exposure.

Identification of the nucleotide exchange factor BmGrpE and its role under high-temperature stress in silkworm, Bombyx mori.

The high-temperature stress gene GrpE plays an important role in coping with high-temperature stress. The mutation of key sites of this gene can improve the high-temperature resistance of organisms. In the present study, using complementary DNAs from the silkworm fat body as the template, the open reading frame sequence of the GrpE gene (BmGrpE) was amplified and was found to be 644 bp in length and encode a protein with a predicted molecular weight of 24.1 kDa. The presence of a binding site for the heat shock transcription factor (Hsf1) at -1440 bp upstream of its coding region indicates that BmGrpE may respond to high-temperature stress. BmGrpE was constitutively expressed throughout developmental stages, with the highest level observed in the 5th instar larvae stage. Moreover, in 5th instar larvae (the 3th day), BmGrpE was expressed in all tissues examined, with the highest levels in the fat body, silk gland, and midgut. Interestingly, under high-temperature stress, TiO2 nanoparticle treatment increased the messenger RNA levels of BmGrpE in the fat body and silk gland. After treatment with dsRNA of BmGrpE, the cell viability of BmN cells was significantly decreased under 34°C and H2 O2 stress (p < .05). Mutation of BmGrpE (H163L) enhanced the resistance of BmN cells under high-temperature stress. These results provide new clues for the study of molecular mechanisms of insect resistance to high temperatures.

Transcriptome analysis reveals gene expression changes of the fat body of silkworm (Bombyx mori L.) in response to selenium treatment.

A comparative transcriptome analysis was conducted to investigate the gene expression changes in the fat body of silkworm after treatment with different concentrations (50μM and 200μM) of selenium (Se). 912 differential expression genes (DEGs) (371 up-regulated and 541 down-regulated) and 1420 DEGs (1078 up-regulated and 342 down-regulated) were identified in silkworm fat body treated with 50μM and 200μM of Se, respectively. In case of 50μM group, DEGs were mainly enriched in the peroxisome pathway and fatty acid metabolism pathway, and later were associated with antioxidant defense and nutrition regulation. After 200μM Se-treatment, DEGs were mainly located in the glycerolipid metabolism and arachidonic acid metabolism pathways, which further encoded detoxification related genes. Furthermore, 32 candidate DEGs from these pathways had been selected to confirm the RNA-seq data. Among these DEGs, 14 genes were up-regulated in the 50μM Se-treated group (only three genes in the 200μM Se-treated group) which were involved in lipid metabolism and antioxidant defense, and 13 up-regulated genes (only two genes were up-regulated in the 50μM Se-treated group) were involved in detoxification of the 200μM Se-treated group. These changes showed that lower concentration of Se could regulate the nutrition and promote antioxidation pathways; whereas, high levels of Se promoted the detoxification of silkworm. These findings can be helpful to understand the possible mechanisms of Se action and detoxification in silkworm and other insects.

Overexpression of BmFoxO inhibited larval growth and promoted glucose synthesis and lipolysis in silkworm.

Forkhead box O (FoxO) is a downstream transcription factor of the insulin-signaling pathway, which plays vital roles in the growth and metabolism of organisms. In this study, BmFoxO was overexpressed in BmE cells, in which proliferation was inhibited and apoptosis was increased. The transgenic vector overexpressing BmFoxO was constructed, and the transgenic silkworm line A4FoxO was generated via embryonic microinjection. The body size of A4FoxO silkworm was smaller than that of non-transgenic silkworm (WT). The quantitative polymerase chain reaction results revealed that the insulin pathway was enhanced and the growth-related TOR pathway was suppressed. Furthermore, the translation of proteins in the fat body of A4FoxO silkworm was inhibited. The expression level of genes involved in the glucose synthesis and lipolysis pathways was increased, whereas that of genes involved in fat synthesis was decreased. Oil red O staining revealed that the amount of lipid droplets was reduced in A4FoxO silkworms compared with WT. Further analysis showed that the content of triglyceride and glycogen was significantly decreased in fat body, but the content of glucose and trehalose was increased in the hemolymph of A4FoxO silkworms. These results suggest that the enhanced expression of BmFoxO disturbs glycolipid metabolism and affects silkworm growth.