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The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a "CTCF-FOX-HOX-SOX" association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated "closed" and "open" conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in silkworms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cotton bollworm, Helicoverpa armigera, is a significant global agricultural pest, particularly detrimental during its larval feeding period. Insects' odorant receptors (ORs) are crucial for their crop-feeding activities, yet a comprehensive analysis of H. armigera ORs has been lacking, and the influence of hormones on ORs remain understudied. Herein, we conducted a genome-wide study and identified 81 ORs, categorized into 15 distinct groups. Analyses of protein motifs and gene structures revealed both conservation within groups and divergence among them. Comparative gene duplication analysis between H. armigera and Bombyx mori highlighted different duplication patterns. We further investigated subcellular localization and protein interactions within the odorant receptor family, providing valuable insights for future functional and interaction studies of ORs. Specifically, we identified that OR48 and OR75 were abundantly expressed during molting/metamorphosis and feeding stages, respectively. We demonstrated that 20E induced the upregulation of OR48 via EcR, while insulin upregulated OR75 expression through InR. Moreover, 20E induced the translocation of OR48 to the cell membrane, mediating its effects. Functional studies involving the knockdown of OR48 and OR75 revealed their roles in metamorphosis development, with OR48 knockdown resulting in delayed pupation and OR75 knockdown leading to premature pupation. OR48 can promote autophagy and apoptosis in fat body, while OR75 can significantly inhibit apoptosis and autophagy. These findings significantly contribute to our understanding of OR function in H. armigera and shed light on potential avenues for pest control strategies., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The silkworm (Bombyx mori) is a model organism for lepidopteran insects. It is an oligophagous insect that primarily feeds on mulberry leaves and has industrial use for the production of raw silk. The development of artificial diets has provided an alternative nutrient source for silkworms; however, one significant issue is that the production of cocoons is lower in silkworms reared on artificial diets compared with those reared on mulberry leaves. The differences in the silk gland in the late-stage fifth instar silkworm larvae, when silk synthesis is most active, between those raised on artificial diets and mulberry leaves, are unknown. In this study, we identified differences in the transcriptomes of the middle and posterior silk glands of fifth instar day five silkworm larvae reared on artificial diets compared with those reared on mulberry leaves using three strains: Daizo, Nichi01, and J137 × C146. We found that the silk-related genes fibrohexamerin (fhx), fibroin-light-chain (fibL), and fibroin-heavy-chain (fibH) in the middle silk gland, and ser1 in the posterior silk gland, were differentially expressed in a strain-dependent manner. In silkworms reared on artificial diets, fhx, fibL, and fibH in the middle silk gland were upregulated in Nichi01 and downregulated in J137 × C146, whereas ser1 in the posterior silk gland was upregulated in J137 × C146 compared with silkworms reared on mulberry leaves. Our results demonstrate that the diet and strain of silkworm larvae affect the expression of genes related to silk production in their silk glands during the late fifth instar stage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shengxiang Zhang reports financial support was provided by China Agriculture Research System of MOF and MARA. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes., (Copyright © 2021. Published by Elsevier B.V.)

Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Juvenile hormone (JH) plays important roles in the control of many biological processes in insects, such as development, reproduction, and polyphenism. JH is primarily produced in the corpora allata (CA) by specific JH biosynthetic enzymes under strict temporal regulation. In a previous study, we identified a novel putative JH biosynthetic gene, protein tyrosine phosphatase, mitochondrial 1 (PTPMT1), from silkworm, Bombyx mori, whose expression is nearly exclusive in the CA and is correlated with JH synthetic activities during late larval development. In this study, to reveal the function of PTPMT1 in vivo, we generated PTPMT1 knockout silkworms using TALEN. In the knockout mutants, no signs indicating defects in JH activity were observed. Instead, PTPMT1 knockout silkworms showed embryonic lethality, developmental arrest, and 3rd-instar lethality not only in mutants lacking total enzymatic activity but also in mutants lacking mitochondrial translocation signals. Moreover, in PTPMT1 knockout embryos, the expression of two genes encoded by the mitochondrial genome, CYTB and ND3, was decreased, indicating a mitochondrial disorder. These results suggested that PTPMT1 plays conserved vital role(s) reported in vertebrates in insect mitochondria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Silkworms are economically important insects because of the value of their silk. After finishing silk spinning, silkworms begin another important physiological process, vitellogenesis. In this study, we explored the relationship between silk spinning and vitellogenin (BmVg) expression in silkworms. In silkworms with the silk fibroin heavy chain gene knocked-out, the concentration of amino acids in the hemolymph was found to be significantly higher than that in the wild type, and the expression of BmVg was advanced at day 7 of the fifth instar stage and 0 h after spinning. Furthermore, through culturing fat body in vitro with different substances treatment including glucose, trehalose, amino acids, 20-hydroxyecdysone, and insulin, we found that only amino acids could induce BmVg expression. RNA interference of BmTOR1 in female silkworms could down-regulate BmVg transcription, resulting in shortened egg ducts and smaller eggs relative to the control. Therefore, these results showed that amino acids could induce BmVg expression through the TOR signaling pathway. Fat body cultured with amino acids in vitro and experiments involving amino acids injected into the silkworm showed that the majority of main amino acids of silk protein could induce BmVg expression. These results suggested that BmVg expression is related to silk spinning and this study would lay a foundation for elucidating the stage specificity expression of BmVg., (Copyright © 2019. Published by Elsevier Ltd.)

Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (BmCDA6, BmCDA7 and BmCDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, BmCDA7 exhibited the highest activity (0.284 μmol/min/μmol), while BmCDA8 showed lower activity of 0.061 μmol/min/μmol. BmCDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6, high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among BmCDA6, BmCDA7 and BmCDA8 proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases participating in tissue remodeling. Three Mmps genes have been identified from the silkworm, Bombyx mori, and their expression levels and enzyme activity are consistent with progressive fat body cell dissociation during the early pupal stages. Using both loss-of-function and gain-of-function experiments, we have demonstrated that Mmps are functionally required for fat body cell dissociation and ovary development in female pupae. Moderate inhibition of Mmps activity via inhibitor treatments delayed fat body cell dissociation and ovary development, while severe inhibition blocked these developmental processes and eventually led to pupal lethality. Individual RNAi knockdown of each Mmp delayed fat body cell dissociation, with the strongest and weakest phenotypes occurring for Mmp3 and Mmp1, respectively. By contrast, overexpression of each Mmp promoted fat body cell dissociation and ovary development, with the strongest stimulatory effects for Mmp3 overexpression and the weakest effects for Mmp1 overexpression. This is the first time to show that Mmps induce fat body cell dissociation in Lepidoptera, and we also hypothesize that Mmps-induced fat body cell dissociation is required for ovary development in this insect species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Heavy-metal-contaminated soil threatens human health and environmental safety. Complete remediation of contaminated soil is expensive; therefore, phytomanagement has emerged as a cost-effective alternative. The current study investigated mulberry tree (Morus alba) plantation, silkworm cultivation, and silk production as phytomanagement options. Results defined the safety of planting mulberry trees in soils with lead (Pb) and arsenic (As) concentrations lower than 369 and 180mgkg -1 , respectively. Silkworms fed with mulberry leaves collected from slightly contaminated soil exhibited productive growth and normal silk production. The silk, silkworm chrysalis, and silkworm fecal matter reached the national standards for textiles, feed, and agricultural sludge, respectively. Based on risk assessment, planting mulberry trees instead of rice significantly decreased the human health risk from contaminated soil. The total carcinogenic risk (Risk d ) and noncarcinogenic risk (HQ d ) derived from mulberry tree plantation and textile production were 2.4×10 -8 and 6.7×10 -5 , respectively, whereas those derived from rice plantation and ingestion were 0.44 and 18.4, respectively. Cost-benefit analysis showed that a mulberry tree plantation can yield $25,675 for every 1ha soil, whereas a rice plantation can yield $8409 for the same area. Moreover, phytoextraction requires $50,000-$150,000 to remediate 1ha of soil. Therefore, constructing a mulberry tree plantation with silkworm culture and silk production is a safe means to utilize slightly contaminated soil., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Insect growth and development are mainly regulated via synchronization of many extrinsic and intrinsic factors such as nutrition and hormones. Previously we have demonstrated that larval growth period influences the effect of insulin on the accumulation of glycogen in the fat body of Bombyx larvae. In the present study we demonstrate that Bombyx larvae at the terminal growth period (TGP, after critical weight) had a significantly greater increase in the expression level of Akt in the fat body than at the active growth period (AGP, before critical weight). The larvae at TGP also showed an increase in the expression level of ecdysone receptors (EcRB1 and USP1) and ecdysone-induced early genes (E75A and broad). The treatment of bovine insulin and methoprene to larvae at AGP induced the transcript levels of Akt, irrespective of the nutritional status of the larvae. However, in larvae at TGP, insulin repressed the transcript level of Akt. On contrary, 20-hydroxyecdysone (20E) induced the expression level of Akt in TGP larvae, but at feeding only. Insulin and 20E thus showed an antagonistic action on the Akt expression level in TGP larvae under feeding. The studies thus showed that larval growth period influences the expression level of Akt and ecdysone receptors in Bombyx. Further, the growth period and nutrition modulate the effect of exogenous hormones on Akt expression., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ommochromes are major pigments involved in coloration of eggs, eyes, wings, and epidermis of insects. Bombyx mori (silkworm) eggs contain a mixture of ommochrome pigments and their precursors. Here, we analyzed the pigment composition of every egg color strain using egg color mutants (w-2, pe, and re) and wild-type strains (dazao and C108) by using full wavelength scanning and high-performance liquid chromatography. We identified ommochrome pigments and their precursors in pigment extracts from non-diapause eggs and diapause eggs, and found that the quantities of ommochrome precursor 3-hydroxy-kynurenine were much higher in the diapause eggs. Ommochrome pigments were absent in the non-diapause eggs. We analyzed the pigment composition of every egg color strain and found an accumulation of 3-hydroxy-kynurenine and absence of ommochromes in the yellow eggs (w-2 and pe), suggesting that the essential factors for ommochrome biosynthesis are high levels of 3-hydroxy-kynurenine, enzymes for ommochrome synthesis and transferase, and spermatiation. Moreover, we confirmed that both decarboxylated xanthommatin and xanthommatin are major ommochrome pigments, and the quantity of decarboxylated xanthommatin is much higher than that of xanthommatin in silkworm eggs. Since ommochrome pigments can change color under oxidative/reductive conditions and the egg color mutant re turns crimson when preserved at a low temperature for a few weeks, we used an oxidation-reduction reaction in vitro to explore mechanisms behind the pigment-based color change. Specifically, during diapause, the contents of decarboxylated xanthommatin and xanthommatin are increased, and the ommochrome pigments convert into their reduced forms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The traditional hybrid breeding techniques can no longer meet the increasing demands for silk production by the silkworm, Bombyx mori, and further improvement of the silk yield will depend on modern molecular breeding techniques. Here, we report improved silk yield in transgenic silkworms overexpressing the oncogene Yorkie CA specifically in the posterior silk gland (PSG). The Yorkie CA cDNA was ligated downstream of the hr3 enhancer and the fibroin L-chain (Fil) promoter, then inserted into a piggyBac vector for transgene. Overexpression of Yorkie CA in the PSG significantly increased the weight of the PSG, and also increased the weight of the cocoon, larval body, and pupal body to decreasing degrees. Overexpression of Yorkie CA up-regulated the Yorkie target genes resulting in increased cell size, endomitosis, the number of protein synthesis organelles, the expression of fibroin genes in the PSG, and eventually silk yield. Additionally, as we reported previously using the binary GAL4/UAS system, transgenic silkworms overexpressing Ras1 CA with the hr3 enhancer and the Fil promoter also showed improved silk yield. Unfortunately, the hybrid progeny of Yorkie CA -overexpressing silkworms and Ras1 CA -overexpressing silkworms did not show overlapping improved silk yield due to the failure to increase expression of both Yorkie and Ras1., (Copyright © 2017. Published by Elsevier Ltd.)

It is widely accepted that the anti-metamorphic action of insect juvenile hormones (JHs) is required to inhibit larval-pupal metamorphosis. However, recent studies using RNAi or knockout techniques reveal that larval status may be maintained independently of JHs during the early larval stages. To investigate why larvae of very early instars do not have competence to metamorphose and how they acquire this competence through larval development, we revisited the classic experiments of Piepho (ca. 1930s) and performed implantation assays using the integument of very young larvae of the silkworm, Bombyx mori. Here, we demonstrate that when the integuments of neonate larvae or newly molted second instar larvae are implanted into last instar host larvae, they are able to directly produce pupal cuticle at the time of pupal metamorphosis of the host. To investigate whether the pupal commitment of implants from the neonate first instar larvae is repressed by JHs, the integuments of Met1 knockout larvae lacking a functional JH receptor were implanted into penultimate instar larvae. We found that the implants of Met1 knockout neonate larvae produced patched pupal cuticles after the host larval molt, whereas those of the wild-type strain produced only larval cuticle without any trace of pupal cuticle. Taken together, our results suggest that the epidermis of very early instar larvae can be pupally committed when provided with unidentified blood-borne factor(s) present in final-instar larvae, and that JHs can block the action of that factor(s) to prolong the feeding period until larvae attain a size appropriate for metamorphosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our previous studies showed that adenosine 5'-monophosphate-activated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the signaling involved in PTTH-stimulated phosphorylation of 4E-BP. We found that 4E-BP phosphorylation stimulated by PTTH was partially reduced in Ca 2+ -free medium, indicating the involvement of Ca 2+ . In addition, we found that a potent and specific inhibitor of phospholipase C (PLC), U73122, greatly inhibited 4E-BP phosphorylation. However, PTTH-stimulated 4E-BP phosphorylation was not attenuated by a protein kinase C (PKC) inhibitor (chelerythrine C). These results indicate that PLC, but not PKC, is involved in PTTH-stimulated 4E-BP phosphorylation. When PGs were treated with agents that directly elevate the intracellular Ca 2+ concentration (either A23187 or thapsigargin), a great increase in 4E-BP phosphorylation was observed. A23187-stimulated phosphorylation of 4E-BP was blocked by a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), but not by U0126, indicating involvement of AMPK and PI3K. Determination of AMPK phosphorylation showed that treatment with either A23187 or thapsigargin inhibited AMPK phosphorylation. Moreover, PTTH appeared to inhibit AMPK phosphorylation in a Ca 2+ -dependent manner. Altogether, these results indicate interconnections among Ca 2+ signaling, AMPK, and 4E-BP phosphorylation in PTTH-activated PGs of B. mori., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In addition to the genome editing technology, novel functional analyses using electroporation are powerful tools to reveal the gene function in the color pattern formation. Using these methods, several genes involved in various larval color pattern formation are clarified in the silkworm Bombyx mori and some Papilio species. Furthermore, the coloration pattern mechanism underlying the longtime mystery of female-limited Batesian mimicry of Papilio polytes has been recently revealed. This review presents the recent progress on the molecular mechanisms and evolutionary process of coloration patterns contributing to various mimicry in Lepidoptera, especially focusing on the gene function in the silkworm and Papilio species., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated-particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A complex signaling network appears to be involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). Less is known about the genomic action of PTTH signaling. In the present study, we investigated the effect of PTTH on the expression of Bombyx mori HR38, an immediate early gene (IEG) identified in insect systems. Our results showed that treatment of B. mori PGs with PTTH in vitro resulted in a rapid increase in HR38 expression. Injection of PTTH into day-5 last instar larvae also greatly increased HR38 expression, verifying the in vitro effect. Cycloheximide did not affect induction of HR38 expression, suggesting that protein synthesis is not required for PTTH's effect. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), partially inhibited PTTH-stimulated HR38 expression, implying the involvement of both the ERK and PI3K signaling pathways. When PGs were treated with agents that directly elevate the intracellular Ca(2+) concentration (either A23187 or thapsigargin), an increase in HR38 expression was also detected, indicating that Ca(2+) is involved in PTTH-stimulated HR38 gene expression. A Western blot analysis showed that PTTH treatment increased the HR38 protein level, and protein levels showed a dramatic increase during the later stages of the last larval instar. Expression of HR38 transcription in response to PTTH appeared to undergo development-specific changes. Treatment with ecdysone in vitro did not affect HR38 expression. However, 20-hydroxyecdysone treatment decreased HR38 expression. Taken together, these results demonstrate that HR38 is a PTTH-stimulated IEG that is, at least in part, induced through Ca(2+)/ERK and PI3K signaling. The present study proposes a potential cross talk mechanism between PTTH and ecdysone signaling to regulate insect development and lays a foundation for a better understanding of the mechanisms of PTTH's actions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

N,O-Carboxymethyl chitosan (NOCC) can prevent postsurgical adhesion formation. Here, we described the preparation of a novel silkworm pupa NOCC and its effects on the prevention of postoperative adhesion in a rat cecal abrasion model. The degree of deacetylation (DDA) of silkworm pupa chitosan was only 49.87 ± 0.86%; regardless, it was used as the raw material to construct the novel silkworm pupa NOCC, which had a weaker crystallinity than the NOCC standard. Sixty male Sprague-Dawley rats were divided into three groups and treated as follows: 0.9% normal saline solution as a negative control, medical anti-adhesion gel as a positive control and the silkworm pupa NOCC anti-adhesion solution. Two and three weeks after surgery, the animals were killed and the adhesion formation was scored. The silkworm pupa NOCC solution significantly decreased the levels of WBC, TNF-α, IL-1β, IL-2, IL-6 and IL-8 but had no effect on IL-4. Additionally, a lower level of TGF-β1 expression was found in the silkworm pupa NOCC group, and significantly less collagen (P<0.01) and fewer inflammatory cells and fibroblasts were detected in the animals of this group. These results suggested that the novel NOCC from silkworm pupa using the method described here have potential applications in the prevention of postoperative intestinal adhesion., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The silkworm Bombyx mori contains high concentrations of free D-serine, an optical isomer of L-serine. To elucidate its function, we first investigated the localization of D-serine in various organs of silkworm larvae, pupae, and adult moths. Using immunohistochemical analysis with an anti-D-serine antibody, we found D-serine in the microvilli of midgut goblet and cylindrical cells and in peripheral matrix components of testicular and ovarian cells. By spectrophotometric analysis, D-serine was also found in the hemolymph and fat body. D-Alanine was not detected in the various organs by immunohistochemistry. Serine racemase, which catalyzes the inter-conversion of L- and D-serine, was found to co-localize with D-serine, and D-serine production from L-serine by intrinsic serine racemase was suggested. O-Phospho-L-serine is an inhibitor of serine racemase, and it was administered to the larvae to reduce the D-serine level. This reagent decreased the midgut caspase-3 level and caused a delay in spermatogenesis and oogenesis. The reagent also decreased mature sperm and egg numbers, suggesting D-serine participation in these processes. D-Serine administration induced an increase in pyruvate levels in testis, midgut, and fat body, indicating conversion of D-serine to pyruvate. On the basis of these results, together with our previous investigation of ATP biosynthesis in testis, we consider the possible involvement of D-serine in ATP synthesis for metamorphosis and reproduction., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Juvenile hormone (JH) synthesis by the corpora allata (CA) does not occur during the pupal stage in both male and female Bombyx mori but begins shortly before adult ecdysis and thereafter only in females. JH biosynthesis in female adults was prevented by allatotropin (AT) through the corpora cardiaca (CC) and the tightly attached oesophagus before adult ecdysis, but after ecdysis, removal of the CC had little effect and only the oesophagus was necessary for AT to prevent JH synthesis. AT could not prevent JH synthesis by the CA alone in either stage. Short neuropeptide F (sNPF) acted directly on the CA, preventing JH biosynthesis without preventing the JH biosynthetic enzymes before adult ecdysis, but had little effect after ecdysis, indicating that the action of AT was not via sNPF. The inhibition of JH synthesis by AT was indirect. Both AT and a factor(s) from the AT-stimulated oesophagus through the CC were necessary for the inhibitory action, which was due to the prevention of some of the JH biosynthetic enzymes. These results clearly show that AT prevents JH synthesis in adult CA in B. mori, although AT stimulates JH biosynthesis by the CA in several insect species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mβ (E(spl)mβ), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mβ was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mβ was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mβ are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Disruption of the appropriate balance between juvenile hormone (JH) and ecdysteroids causes abnormal insect development. The application of a JH analog (JHA) during the early days of the final (fifth) instar induces dauer larvae with low ecdysteroid titers in insects, but the mechanism that underlies the action of JHA remains unclear. In this study, we clarified the negative effects of JHA on ecdysteroidogenic enzymes. JHA application to Bombyx mori larvae during the early stage of the fifth instar suppressed the expression of four enzymes, i.e., neverland (nvd), spook, phantom, and disembodied but not non-molting glossy and shadow. Furthermore, JHA application reduced the amount of 7-dehydrocholesterol, a metabolite produced by Nvd, in both the prothoracic glands and hemolymph, indicating JHA can disrupt ecdysteroidogenic pathway from the first step. Neck ligation resulted in increased nvd expression, whereas JHA application reversed this increase. These results suggest that the endogenous JH represses ecdysteroidogenesis during the early days in final instar larvae. Neck ligation and JHA application had no substantial effects on the expression of a transcription factor, ftz-f1, or a prothoracicotropic hormone receptor, torso; therefore, the inhibitory regulation of JHA may not involve these factors. Further analysis is required to clarify the regulation of JHA in ecdysteroidogenesis, but this study showed that JHA, and probably endogenous JH, can suppress the transcription of four of six ecdysteroidogenic enzymes. This regulation may be essential for maintaining the appropriate balance between JH and ecdysone during insect development., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -β, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Haemocytes play crucial roles in immune responses and survival in insects. Specific cell markers have proven effective in clarifying the function and haematopoiesis of haemocytes. The silkworm Bombyx mori is a good model for studying insect haemocytes; however, little is known about haemocyte-specific markers or their functions in silkworm. In this study, we identified the α subunit of integrin, BmintegrinαPS3, as being specifically and highly expressed in silkworm haemocytes. Immunofluorescence analysis validated the specificity of BmintegrinαPS3 in larval granulocytes. Further analyses indicated that haemocytes dispersed from haematopoietic organs (HPOs) into the circulating haemolymph could differentiate into granulocytes. In addition, the processes of encapsulation and phagocytosis were controlled by larval granulocytes. Our work demonstrated that BmintegrinαPS3 could be used as a specific marker for granulocytes and could be applied to future molecular cell biology studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The silkworm, Bombyx mori, is an important model of lepidoptera insect, and it has been used for several models of human diseases. In human being, long-term high-sugar diet can induce the occurrence of diabetes and other related diseases. Interestingly, our experiments revealed the high glucose diet also has a suppressive effect on the development of silkworms. To investigate the molecular mechanism by which high-glucose diet inhibited the midgut growth in silkworms, we employed comparative proteomic analysis to globally identify proteins differentially expressed in normal and high-glucose diet group silkworms. In all, 28 differently proteins were suppressed and 5 proteins induced in high-glucose diet group. Gene ontology analysis showed that most of these differently proteins are mainly involved in metabolic process, catalytic and cellular process. A development related protein, imaginal disk growth factor (IDGF), was further confirmed by western blot exclusively expressing in the normal diet group silkworms. Taken together, our data suggests that IDGF plays a critical role in impairing the development of silkworms by a high-glucose diet., Biological Significance: Glucose has been thought to play essential roles in growth and development of silkworm. In this paper, we certified firstly that high-glucose diet can suppress the growth of silkworm, and comparative proteomic was employed to reveal the inhibition mechanism. Moreover, an important regulation related protein (IDGF) was found to involve in this inhibition process. These results will help us get a deeper understanding of the relationship between diet and healthy. Furthermore, IDGF may be the critical protein for reducing the blood sugar in silkworm, and it may be used for screening human hypoglycemic drug. The work has not been submitted elsewhere for publication, in whole or in part, and all the authors have approved the manuscript., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although the pupae and larvae of Bombyx mori possess especially large amounts of free d-serine, the physiological role of the amino acid in the silkworm is unknown. We investigated the effect of d-serine on spermatogenesis. A lowered d-serine level throughout larval development caused a delay in spermatogenesis and resulted in reduced numbers of eupyrene sperm. Administration of d-serine transiently increased the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2; hereafter, ERK) by approximately 25% in the testis of day 3 fifth instar larvae. l-Serine had no effect on ERK activation, and other organs did not respond to d-serine. The effect of d-serine on ERK activation was confirmed by administering d-serine dehydratase, an enzyme that specifically degrades d-serine, and the enzyme's inhibitor, hydroxylamine. ERK phosphorylation in the testis was significantly inhibited by Go6983 and U0126, inhibitors of protein kinase C (PKC) and mitogen-associated protein kinase kinase 1/2 (MEK), respectively, but not by H-89, a protein kinase A (PKA) inhibitor, indicating that ERK was activated in the testis via PKC and MEK but not via PKA. The inhibition of ERK phosphorylation by Go6983 or U0126 was reduced by 20-30% by d-serine. Roughly 30% of c-Raf phosphorylation at an inhibitory site (Ser259) was decreased by the addition of d-serine. These results suggest that d-serine activates ERK in the testis of silkworms through a pathway including c-Raf but not PKC or MEK. Immunohistochemistry confirmed d-serine-induced ERK phosphorylation in the testis and revealed the presence of phospho-ERK in the nuclei of spermatocytes and spermatids., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Injection or feeding of dsRNA is commonly used to induce specific gene silencing by RNAi in insects but very little research has been carried out to investigate non-specific effects on gene expression of dsRNA as pathogen-associated molecular pattern (PAMP). This study focuses on the potential role of the BmToll9-1 receptor to modulate the transcriptional response of innate immune and RNAi genes to dsRNA and lipopolysaccharide (LPS), which was used for comparison. To study this role, we took advantage of the silkmoth-derived Bm5 cell line, which does not express BmToll9-1 endogenously, and engineered a transformed cell line that permanently expresses BmToll9-1. Quantitative mRNA expression studies showed that BmToll9-1 can significantly alter the transcriptional response to dsRNA and LPS: (1) BmToll9-1 promotes the transcriptional response of Dicer2, encoding a key component of the RNAi machinery, and, to a lesser extent, that of transcription factors in the Jak-STAT and Toll pathways; and (2) BmToll9-1 represses the transcriptional induction of the IMD and Jak-STAT pathway genes, as well as the antimicrobial peptide (AMP) effector genes, by LPS. Thus, BmToll9-1 was identified as a modulator of innate immune and RNAi machinery gene expression that could be related to its preferential expression in the larval gut, the major barrier of pathogen entry. While BmToll9-1 was found to modulate RNAi-related gene expression, a reporter-based RNAi assay established no evidence for a direct interaction of BmToll9-1 with the intracellular RNAi machinery., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Our previous study demonstrated that mitochondria-derived reactive oxygen species (ROS) generation is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the mechanism of ROS production and the signaling pathway mediated by ROS. PTTH-stimulated ROS production was markedly attenuated in a Ca(2+)-free medium. The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated ROS production, indicating the involvement of Ca(2+) and PLC. When the PGs were treated with agents that directly elevate the intracellular Ca(2+) concentration (either A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)), a great increase in ROS production was observed. We further investigated the action mechanism of PTTH-stimulated ROS signaling. Results showed that in the presence of either an antioxidant (N-acetylcysteine, NAC), or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)), PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK was blocked. Treatment with 1mM of H2O2 alone activated the phosphorylation of ERK and 4E-BP, and inhibited AMPK phosphorylation. From these results, we conclude that PTTH-stimulated ROS signaling is Ca(2+)- and PLC-dependent and that ROS signaling appears to lie upstream of the phosphorylation of ERK, 4E-BP, and AMPK., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sexual differentiation in the silkworm Bombyx mori is controlled by sex-specific splicing of Bmdsx, in which exons 3 and 4 are skipped in males. B. mori insulin-like growth factor II mRNA-binding protein (Imp) is a factor involved in the male-specific splicing of Bmdsx. In this study, we found that the male-specific Imp mRNA is formed as a result of the inclusion of exon 8 and the promoter-distal poly(A) site choice, whereas non-sex-specific polyadenylation occurs at the promoter-proximal poly(A) site downstream of exon 7. Recent studies revealed that Drosophila Sxl, tra in several dipteran and hymenopteran insects, and fem in Apis mellifera, play a central role in sex determination and maintain their productive mode of expression via an autoregulatory function. To determine whether Imp protein is required for the maintenance of the male-specific splicing of its own pre-mRNA, we knocked down endogenous Imp in male cells and assessed the male-specific splicing of an exogenous Imp minigene. Knockdown of endogenous Imp inhibited the male-specific splicing of the Imp minigene transcript. In contrast, overexpression of Imp in female cells induced the male-specific splicing of the Imp minigene transcript. Moreover, deletion of adenine-rich (A-rich) sequences located downstream of the proximal poly(A) site repressed the male-specific splicing of the Imp minigene transcript. Finally, gel shift analysis demonstrated that Imp binds to the A-rich sequences. These data suggest that Imp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Lipid transfer particle (LTP) is a high-molecular-weight, very high-density lipoprotein known to catalyze the transfer of lipids between a variety of lipoproteins, including both insects and vertebrates. Studying the biosynthesis and regulation pathways of LTP in detail has not been possible due to a lack of information regarding the apoproteins. Here, we sequenced the cDNA and deduced amino acid sequences for three apoproteins of LTP from the silkworm (Bombyx mori). The three subunit proteins of the LTP are coded by two genes, apoLTP-II/I and apoLTP-III. ApoLTP-I and apoLTP-II are predicted to be generated by posttranslational cleavage of the precursor protein, apoLTP-II/I. Clusters of amphipathic secondary structure within apoLTP-II/I are similar to Homo sapiens apolipoprotein B (apoB) and insect lipophorins. The apoLTP-II/I gene is a novel member of the apoB/large lipid transfer protein gene family. ApoLTP-III has a putative conserved juvenile hormone-binding protein superfamily domain. Expression of apoLTP-II/I and apoLTP-III genes was synchronized and both genes were primarily expressed in the fat body at the stage corresponding to increased lipid transport needs. We are now in a position to study in detail the physiological role of LTP and its biosynthesis and assembly.

Chemosensory proteins (CSPs) are a family of small soluble proteins that, in addition to the odorant-binding proteins (OBPs), are involved in chemical communication. To understand the physiological function of the 16 known CSPs in the silkworm Bombyx mori, we investigated the expression patterns in different tissues and developmental stages using quantitative real-time RT-PCR (Q-PCR) and Western blot analysis. The results indicated that most CSPs were widely expressed in embryos, larvae, pupae and adults but were developmentally regulated. Such broad spatial and temporal expression was inconsistent with a specific association with chemosensory function. We conclude that CSPs are multifunctional proteins that are involved in diverse cellular processes and that can play non-chemosensory as well as chemosensory roles. Binding experiments revealed different binding characteristics of CSP1 and CSP2, with retinal being the best ligand, suggesting a putative function of these CSPs as carriers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hemocytes play multiple important roles during insect growth and development. Five types of hemocytes have been identified in the silkworm, Bombyx mori: prohemocyte, plasmatocyte, granulocyte, spherulocyte, and oenocytoid. We used the S-phase marker bromodeoxyuridine (BrdU) antibody along with the mitosis marker phosphohistone H3 (PHH3) antibody to monitor proliferation of hemocytes in vivo. The results indicate that silkworm hematopoiesis not only occurs in the circulatory system but also in hematopoietic organs (HPOs). During the 5th instar, the hemocyte proliferation in the circulatory system reaches a peak at the pre-wandering stage. Following infection by Escherichia coli, circulating hemocytes increase their cell divisions as demanded by the cellular immune response. All hemocytes, except spherulocytes, have the capacity to multiply in vivo. The BrdU label-retaining assay shows that a small portion of cells from the circulatory system and the HPOs are continuously labelled up to 9days and 4days respectively. A small number of long-term label retaining cells (LRCs) quiescently locate in circulatory system. All results indicate that there are a few quiescent stem cells or some progenitors in the larval circulatory system and HPO that produce new hemocytes and continuously release them into the circulating system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Injection of dsRNA is widely applied to silence endogenous genes and study gene function in insects. However, it is not yet clear to what extent it can also exert non-specific effects, for instance by interference with the innate immune response. In this study, we report on the transcriptional response of BmToll9-1 to lipopolysaccharide (LPS) and dsRNA in the silkmoth, Bombyx mori. BmToll9-1 encodes a Toll receptor highly expressed in midgut tissue and that shows limited similarity to the mammalian TLR3 endolysosome receptor for dsRNA; while Dcr2 and Ago2 encode two key components of the RNAi machinery. An expression pattern study of all 14 Toll receptors in B. mori showed that BmToll9-1 was expressed in different larval and pupal tissues with the highest expression level detected in the midgut, indicating a possible function in immunity against pathogens taken up by the food. In order to investigate the response of BmToll9-1, different ways to deliver dsRNA, specific for GFP (dsGFP), and LPS were applied in Bombyx 5th instar larvae. The feeding experiments suggested that dsGFP did not suppress the expression of BmToll9-1 significantly, while LPS could suppress the expression of BmToll9-1 after 3h of feeding. On the other hand, the injection experiments showed that dsGFP, as well as LPS, could significantly inhibit the expression of BmToll9-1 in 3h. Bacteria that constantly expressed dsGFP could also down-regulate the expression of BmToll9-1 to a greater extent than bacteria that do not express dsGFP. The failure of dsGFP by feeding to affect the expression of BmToll9-1 was correlated with the rapid degradation of dsGFP by dsRNase in the midgut juice. Expression of the RNAi machinery genes Dcr2 and Ago2, as well as dsRNase, was also affected by injection of dsRNA and not by feeding, but in these cases an induction was observed instead of a down-regulation. Because LPS is a well-known pathogen-associated molecular pattern (PAMP), it suggested that the decrease in BmToll9-1 expression is a consequence of the activation of the innate immune response by LPS. The similar response of BmToll9-1 between the two triggers, LPS and dsRNA, suggests that dsRNA can also act as a PAMP in the midgut of Bombyx. Furthermore, induction of the genes Dcr2, Ago2 and dsRNase may also constitute a defense mechanism against invading dsRNA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms, causing severe economic losses in sericulture. To create antiviral silkworm strains, we constructed a transgenic vector in which the dsRNA for five tandem BmNPV genes was controlled by the BmNPV hr3 enhancer and IE1 promoter. The antivirus gene Bmlipase-1 was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. After infection with a high dose of BmNPV, the survival rates of SW-H and non-transgenic SW were 64% and 13%, respectively. SW-H could be the first transgenic animal that is highly antiviral and that might inhibit the virus at multiple stages of infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

During larval-pupal transformation, the anterior silk glands (ASGs) of the silkworm Bombyx mori undergo programmed cell death (PCD) triggered by 20-hydroxyecdysone (20E). Under standard in vitro culture conditions (0.3 ml of medium with 1 μM 20E), ASGs of the fourth-instar larvae do not undergo PCD in response to 20E. Similarly, larvae of the fifth instar do not respond to 20E through day 5 of the instar (V5). However, ASGs of V6 die when challenged by 20E, indicating that the glands might be destined to die before V6 but that a death commitment is not yet present. When we increased the volume of culture medium for one gland from 0.3 to 9 ml, V5 ASGs underwent PCD. We examined the response of ASGs to 20E every day by culturing them in 9 ml of medium and found that ASGs on and after V2 were fully responsive to 20E. Because pupal commitment is associated with juvenile hormone (JH), the corpora allata (a JH secretory organ) were removed on day 3 of the fourth larval instar (IV3), and their ASGs on V0 were cultured with 20E. Removal of the corpora allata allowed the V0 larval ASGs to respond to 20E with PCD. In contrast, topical application of a JH analogue inhibited the response to 20E when applied on or before V5. We conclude that the acquisition of responsiveness to 20E precedes the loss of JH sensitivity, and that the death commitment in ASGs occurs between V5 and 6., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The K(m) of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The K(m) of bmST1 for xanthurenic acid is low, in the μM range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Blood sugar is an essential energy source for growth and development and is maintained at a constant level through precise regulation of formation and utilization. Sugars are produced from dietary carbohydrates by enzymatic hydrolysis in the digestive tract, which are under the homeostatic control of paracrine and prandial mechanisms in mammals. Here, we show that dietary carbohydrates hydrolyzing activity of the digestive tract is developmentally regulated by the steroid hormone ecdysone in the silkworm, Bombyx mori. The dietary carbohydrates hydrolyzing activity remained high throughout the last larval period and then decreased to negligible levels until the pupal period. However, dietary carbohydrates digestive activities were constitutively high when the steroidogenic organ, prothoracic glands were ablated. The prothoracic glands produced and released a large amount of ecdysone at the end of the larval period, suggesting that ecdysone is responsible for the decrease in dietary carbohydrates hydrolyzing activity. In fact, ecdysone decreased the activity to negligible levels in silkworms lacking the prothoracic glands. The present results indicate that the dietary carbohydrates hydrolyzing activity is regulated by ecdysone and that an increase in ecdysone titer decreases that activity at the end of the larval period, suggesting that ecdysone is essential for metabolic coordination during development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many moths use sex pheromones to find their mates in the dark. Their antennae are well developed with lateral branches to receive the pheromone efficiently. However, how these structures have evolved remains elusive, because the mechanism of development of these antennae has not been studied at a molecular level. To elucidate the developmental mechanism of this type of antenna, we observed morphogenesis, cell proliferation, cell death and antennal patterning gene expression in the branched antenna of the silk moth, Bombyx mori. Region-specific cell proliferation and almost ubiquitous apoptosis occur during early pupal stages and appear to shape the lateral branch cooperatively. Antennal patterning genes are expressed in a pattern largely conserved among insects with branchless antennae until the late 5th larval instar but most of them change their expression dramatically to a pattern prefiguring the lateral branch during metamorphosis. These findings imply that although antennal primordium is patterned by conserved mechanisms before metamorphosis, most of the antennal patterning genes are reused to form the lateral branch during metamorphosis. We propose that the acquisition of a new regulatory circuit of antennal patterning genes may have been an important event during evolution of the sensory antenna with lateral branches in the Lepidoptera., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Diapause hormone (DH) targets developing ovaries in female pupae to induce embryonic diapause immediately after completion of mesoderm segregation of the silkworm, Bombyx mori. At the same time, DH enhances trehalase activity on the oolemma, which leads to higher concentrations of glycogen in oocytes through the stimulated incorporation of hemolymph trehalose. In B. mori, the treh-1 and -2 genes encoding soluble trehalase (68 kDa) and integral-membrane trehalase (74kDa) have been isolated. DH stimulates mRNA expression of both of these genes. In this study, we aimed to clarify whether ovarian trehalase originates from Treh-1 or Treh-2. Western blotting of the developing ovaries showed positive bands in the membrane-bound fraction, containing trehalase activity, only with antibodies against Treh-1&2 and Treh-2, but not Treh-1, irrespective of nondiapause or diapause egg-producers. The intensities of the positively stained 74 kDa bands were increased approximately 4-fold in ovaries from pupae with intact subesophageal ganglion (SG, a unique DH-biosynthesizing organ), and from pupae that were injected with DH at the middle pupal stage after their SGs were removed on the day of pupation. Furthermore, quantitative real-time PCR data showed that in developing ovaries, copy number of treh-2 mRNA per one copy of rp49 mRNA was approximately 1000-fold higher than that of treh-1 mRNA. These results demonstrate that trehalase activities enhanced by DH originate mainly from treh-2 protein regulated at the transcriptional level., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Genetic studies in the fruitfly, Drosophila melanogaster, have uncovered a conserved insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects. From the silkworm, Bombyx mori, we have identified and characterized several key genes involved in the IIS pathway, including InR, IRS, PI3K110, PI3K60, PTEN, PDK, and Akt. Tissue distribution analysis showed that most of these genes were highly expressed in the fat body implying that the IIS pathway is functionally important within insect adipose tissue. Developmental profile studies revealed that the expression levels of InR, IRS, PI3K110, and PDK were elevated in the fat body during molting and pupation, periods when animals ceased feeding and hemolymph levels of 20-hydroxyecdysone (20E) were high. Starvation rapidly up-regulated the mRNA levels of these same genes in the fat body, while 20E slowly induced their transcription. We conclude that 20E slowly reduces food consumption and then indirectly induces a state of starvation resulting in the elevation of the mRNA levels of InR, IRS, PI3K110, and PDK in the Bombyx fat body during molting and pupation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention., (Copyright 2010 Elsevier Ltd. All rights reserved.)