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The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms., Competing Interests: Declaration of competing interest The authors that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bombyx mori is an insect species of great economic importance, and its silk gland is a vital organ for the synthesis and secretion of silk protein. However, long-term artificial domestication of B. mori has resulted in high sensitivity to chemical toxins, especially insecticides. Cyantraniliprole (Cya), a second-generation ryanodine receptor modulator insecticide, is widely utilized in agriculture for pest control. In this study, the impact of Cya toxicity on the development of silk glands in the 5th instar larvae of B. mori was assessed using Cya LC 5 , LC 10 and LC 20 , as well as a starvation treatment group for comparison. Short-term exposure (24 h) to different concentrations of Cya resulted in delayed development of silk glands in B. mori. Meanwhile, the body weight, silk gland weight, silk gland index and cocoon quality were significantly reduced in a concentration-dependent manner, except for the Cya LC 5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC 10 induced disruption of the nuclear membrane and endoplasmic reticulum in the posterior silk gland (PSG) cells, leading to the formation of intracellular vacuoles. Transcriptome sequencing of PSGs identified 2152 genes that were differentially expressed after exposure to Cya LC 10 , with 1153 down-regulated genes and 999 up-regulated genes. All differentially expressed genes were subjected to functional annotation using gene ontology and Kyoto encyclopedia of genes and genomes database, and it was found that protein synthesis-related pathways were significantly enriched, with the majority of genes being down-regulated. Furthermore, the transcription levels of genes involved in "protein processing in endoplasmic reticulum", "protein export", "proteasome" and "DNA replication" were quantified using qRT-PCR. Our findings suggested that short-term exposure to Cya LC 10 resulted in disruption of DNA replication, as well as protein transport, processing and hydrolysis in the PSG cells of B. mori. The results of this study provide a theoretical foundation for the safe utilization of Cya in sericulture production., 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. Published by Elsevier Inc.)

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmβGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmβGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway is highly conserved in mammals, but the pattern recognition receptors (PRRs) and their functions are unclear. We found that the expression pattern of Bombyx mori C-type lectin 5 (BmCTL 5) had a synergy relevance with the JAK/STAT signaling pathway against Beauveria bassiana. An RNAi assay, subcellular localization analysis, yeast two-hybrid technique, protein recruitment experiment and pathogen infection tests were used to explore the roles of BmCTL 5 in the JAK/STAT signaling pathway. Knock-down of the BmCTL 5 suppressed the JAK/STAT signaling pathway and the PO cascade of nodule melanization. BmCTL 5 is located in the cytomembrane and interacted with BmHOP both in yeast and B. mori ovary cells N (BmN cells). BmCTL 5 and the JAK/STAT signaling pathway was activated by B. bassiana but only slightly activated by B. mori cytoplasmic polyhedrosis virus (BmCPV), Nosema bombycis and bacteria LPS. These findings suggest that BmCTL 5 might be an important PRR for the JAK/STAT signaling pathway and may mediate the nodule melanization for fungi infection. These data provide insights into the immune mechanism of the JAK/STAT signaling pathway in insects and aid understanding of the mechanism of the JAK/STAT signaling pathway and adaptive immune systems in mammals., (Copyright © 2020. Published by Elsevier Inc.)

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is known to replicate in many tissues of Bombyx mori larvae. However, the cell lines used for BmNPV research are predominantly derived from B. mori ovaries or early embryos. In the present study, we examined the properties of NIAS-Bm-aff3 (aff3), a cell line that was established from B. mori larval fat body, which is one of the major tissues for BmNPV propagation. aff3 is a floating cell line, and cell adhesion was enhanced following the coating of the culture dish with poly-d-lysine. RT-qPCR assays demonstrated that the expression of germ cell markers, Vasa, Siwi, and BmAgo3, was much lower in aff3 cells as compared to the B. mori ovary-derived cell line BmN-4. Conversely, aff3 cells express an adipocyte marker, Fabp1, at higher levels, indicating that this cell line retains the characteristics of fat body cells. BmNPV infection induces unique cell fusion in aff3 cells, which was also observed following infection with Autographa californica multiple nucleopolyhedrovirus, a virus that does not cause productive infection in B. mori cells. Occlusion bodies (OBs) produced in BmNPV-infected aff3 cells exhibit large cuboidal shapes as compared to those produced in BmN-4 cells. Furthermore, extremely large OBs (~25 μm in side length) were produced in aff3 cells when infected with a cuboidal polyhedrin mutant. Taking into account these unusual properties, we conclude that aff3 could prove to be a useful resource for conducting baculovirus research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The resistance of silkworm to infection by Bombyx mori nuclear polyhedrosis virus (BmNPV) is a main focus of sericultural research. Previously, a BmNPV-resistant strain, NB, was identified among a collection of Chinese silkworm strains in our lab. To better understand the molecular mechanism of NB strain resistance, the patterns of host immune response gene transcription in resistant (NB) and susceptible (306) strains were examined. Quantative real-time PCR (qRT-PCR) revealed that multiple insect innate immune signaling pathways (Toll, Imd and JAK/STAT) were strongly activated upon infection with BmNPV. Notably, Suppressor of cytokine signaling 2 (BmSOCS2) mRNA expression was significantly up-regulated in midgut tissues of the resistant NB strain, suggesting that the BmSOCS2 gene product may be involved in host immune defense against BmNPV infection. A significant inhibition of BmNPV replication was also observed in BmN cells transfected with a vector encoding BmSOCS2. The results suggest that BmSOCS2 is a key gene involved in the resistance of the NB silkworm strain to BmNPV infection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is a severe pathogen for the domestic silkworm, Bombyx mori. BmNPV harbors over 140 protein-coding genes in its 128.4 kilobase pair-long double-stranded genome. However, many BmNPV genes are still uncharacterized. Here we investigated the role of BmNPV Bm96 in both B. mori cultured cells and larvae. We found that Bm96 is mainly expressed at the late stage of infection and accumulation of Bm96 protein peaks at 24 h post infection (hpi) and declines gradually at 48 hpi in B. mori cultured cells. Compared with the wild-type viruses, Bm96-deletion viruses exhibited higher viral propagation and fast-killing phenotype in B. mori larvae. These results strongly suggest that Bm96 negatively regulates the propagation of BmNPV in B. mori larvae. Furthermore, we observed that larvae infected with Bm96-deletion viruses showed lower locomotory activity at the late stage of infection compared with those infected with the wild-type viruses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Beauveria pseudobassiana has great potential for use in the management of various insect pests. In the present study, we aimed to explore the the virulence of B. pseudobassiana isolated from a diversity of hosts to Bombyx mori and Tenebrio molitor larvae. To this end, 15B. pseudobassiana isolates from 10 different geographical locations were identified based on morphological characteristics and molecular data. The phylogenetic positions of the isolates were evaluated according to morphological features and phylogenetic inferences based on six loci (nrSSU, nrLSU, TEF, RPB1, RPB2 and Bloc). In addition to growth in soil, the B. pseudobassiana isolates in our study were isolated from a wide host range that extended to 5 orders, 11 families, and 14 species. Moreover, anamorphically typified B. pseudobassiana was grown for the first time from teleomorph stromata. Pathogenicity of the B. pseudobassiana isolates from the different hosts was determined with two bioassays using B. mori and T. molitor larvae. The results indicated that mortality of B. mori caused by the lepidopteran isolates was significantly higher than that of isolates from other hosts, and virulence of the coleopteran isolates to T. molitor was significantly higher than that of isolates from other hosts. The host specificity of B. pseudobassiana should be studied in more detail before future consideration of isolates for use in biological control of pests., 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.)

Endothiapepsin is an aspartic proteinase that was first isolated from the plant pathogenic fungus Endothia parasitica. In previous studies, we reported on three endothiapepsin-like proteins in the entomopathogenic fungus Beauveria bassiana; the genes were up-regulated in B. bassiana hyper-virulent strain GXsk1011 at early stage infection in the silkworm. However, whether these proteins play a role in pathogenicity or not remains unknown. In this study, we cloned one protein, BbepnL-1 gene (BBA-07766), that has 98% homology with B. bassiana strain Bb2860, and expressed it in the yeast Pichia pastoris to investigate its function. The endothiapepsin-like protein is a secreted proteinase of molecular weight approximately 40 kDa. It has an N-glycosylation site and a mutation in the C-terminal conserved domain- a Thr was mutated to Gly in B. bassiana GXsk1011 and is different than the endothiapepsin of Endothia parasitica. The recombinant endothiapepsin-like protein showed enzyme activity and degraded the protein components of the silkworm cuticle. To further investigate the activity of the endothiapepsin-like protein, we knocked out the gene BbepnL-1 and showed that the loss of BbepnL-1 reduced the virulence in the silkworm. These results demonstrated that the endothiapepsin-like protein of B. bassiana is a virulence factor., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nosema bombycis is an obligate intracellular pathogen that can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Here we investigated the proliferation characteristics of N. bombycis in silkworm embryos using a histopathological approach and deep RNA sequencing. We found that N. bombycis proliferated mainly around yolk granules at the early stage of the embryonic development, 1-2 days post oviposition (dpo). At 4-6 dpo, a portion of N. bombycis in different stages adjacent to the embryo were packaged into the newly formed intestinal lumen, while the remaining parasites continued to proliferate around yolk granules. In the newly hatched larvae (9 dpo), the newly formed spores accumulated in the gut lumen and immediately were released into the environment via the faeces. Transcriptional profiling of N. bombycis further confirmed multiplication of N. bombycis throughout every stage of embryonic development. Additionally, the increased transcriptional level of spore wall proteins and polar tube proteins from 4 dpo indicated an active formation of mature spores. Taken together, our results have provided a characterization of the proliferation of this intracellular microsporidian pathogen in congenitally infected embryos leading to vertical transmission., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hippo signaling pathway (signaling pathway Hippo, hereinafter referred to as the Hippo pathway) was the earliest found in Drosophila (Schneck [1]), which can regulate the development of tissues and organs, even some components of the pathway were identified as tumor suppressor [2]. The pathway was more concerned in fruit flies and mice (Schneck [1]), but little attention has been given in silkworm, an important economic and lepidopteran model insect. In this manuscript, we identified major Hippo pathway related genes (Hippo, Salvador, Warts, Mats, Yorkie) in silkworm and named BmHpo, BmSav, BmWts, BmMats, BmYki. The domain organization of these genes was highly conserved in silkworm and other organisms suggesting that they could use similar protein-protein interactions to construct the Hippo kinase cascades. The expression profiles of these genes in silkworm during embryonic, larval, wandering, pupal and adult stages were analyzed, 14 tissues/organs of the day 3, 5th instar larvae (L5D3) as well. Experimental results showed that the expression of Hippo pathway had some influence on the development of silkworm. In order to find out the mechanism of Hippo pathway affecting silkworm development, BmHpo and BmYki were up-regulated and de-regulated in the cell line of Bombyx mori-BmN-SWU1(NS), and the changes of cell proliferation activity and cell cycle were detected. The distribution of BmYki was detected by immunofluorescence technique. This study provides insights into the genes of Hippo pathway which have a certain effect on somatic development and cell proliferation in silkworm., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Short-chain insecticidal neurotoxin Tx4(6-1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6-1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6-1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6-1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6-1) was purified from a 400 ml fed-batch culture supernatant by Ni + -nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6-1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6-1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5-8.5 μg/g body weight. Dietary toxicity was not observed in silkworm larvae., (Copyright © 2018. Published by Elsevier Inc.)

Insulin and ecdysone signaling play a critical role on the growth and development of insects including Bombyx mori. Our previous study showed that Bombyx larvae reached critical weight for metamorphosis between day 3.5 and 4 of the fifth larval instar. The present study showed that the effect of insulin on the accumulation of glycogen in fat body of Bombyx larvae depends on the critical growth period. When larvae are in active growth period (before reaching critical weight), insulin caused increased accumulation of glycogen, while its treatment in larvae at terminal growth period (after critical period) resulted in an increased mobilization of glycogen. During terminal growth period, insulin and 20-hydroxyecdysone (20E) showed an antagonistic effect on the accumulation of fat body glycogen in fed, food deprived and decapitated larvae as well as in isolated abdomens. Insulin treatment decreased the glycogen content, whereas, 20E increased it. Food deprivation and decapitation caused an increase in the transcript levels of insulin receptor (InR) and this increase in InR expression might be attributed to a decrease in synthesis/secretion of insulin-like peptides, as insulin treatment in these larvae showed a down-regulation in InR expression. However, insulin showed an up-regulation in InR in isolated abdomens and it suggests that in food deprived and decapitated larvae, the exogenous insulin may interact with some head and/or thoracic factors in modulating the expression of InR. Moreover, in fed larvae, insulin-mediated increase in InR expression indicates that its regulation by insulin-like peptides also depends on the nutritional status of the larvae. The treatment of 20E in fed larvae showed an antagonistic effect on the transcript levels since a down-regulation in InR expression was observed. 20E treatment also led to a decreased expression of InR in food deprived and decapitated larvae as well as in isolated abdomens. Insulin and 20E also modulated the expression level of ecdysone receptors (EcRB1 and USP1). 20E treatment showed an up-regulation in expression of ecdysone receptors, but only in fed larvae, whereas insulin treatment showed a down-regulation in the expression of EcRB1 and USP1 in all the experimental larvae studied. Further, the data indicates that an up-regulation of ecdysone receptors is associated with an increase in fat body glycogen content, whereas an up-regulation of insulin receptor expression causes glycogen mobilization. The study, therefore, suggests that the insulin and ecdysone signaling are linked to each other and that both insulin and ecdysone are involved in regulating the carbohydrate reserves in B. mori., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Several lines of evidence have shown that the deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the nucleopolyhedrovirus (NPV) genome increases the killing speed of host lepidopteran larvae. However, it has not been investigated in detail whether the effects of egt deletion depend on the larval stages of the host insect. In this study, we performed bioassays using 10 continuous larval stages of the 4th- or 5th-instar Bombyx mori larvae and B. mori NPV egt mutants. The fast-killing phenotype was observed in the egt mutants only when the infection process progressed through larval-larval transition. All day-2 4th-instar larvae infected with the egt mutants entered the molting stage and died much earlier than wild-type-infected larvae. Bodies of egt mutant-infected larvae were filled with excessive fluid immediately after head capsule slippage, owing presumably to the degeneration of Malpighian tubules. Fourth- or 5th-instar larvae infected with the egt mutants at early stages of each instar died similarly to those infected with the wild-type virus. Under infection in the middle stages of the 5th-instar, the survival time of egt mutant-infected larvae was significantly longer than that of the wild-type virus-infected larvae. These results clearly show that the effects of egt deletion on killing speed of NPV are largely dependent on the developmental stage of the host larvae infected by the virus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The vertebrate estrogens include 17-β-estradiol (E2), which has an analog in silkworm ovaries. In this study, the Bombyx mori vitellogenin gene (BmVg) was used as a biomarker to analyze the function of the E2 in silkworm. In most oviparous animals, Vg has female-specific expression. However, BmVg expression was also detected in B. mori males. Stage specific fluctuation of BmVg expression was similar in males and females, but expression levels in males were lower than in females. E2 treatment by injection or feeding of male larvae in the final instar stage induced and stimulated male BmVg transcription and protein synthesis. When silkworm ovary primordia were transplanted into males, BmVg was induced in male fat bodies. Transplanted ovaries primordia were also able to develop into ovaries and produce mature eggs. When females were treated with E2 promoted BmVg/BmVn protein accumulation in hemolymph, ovaries and eggs. However, BmVg transcription was decreased in female fat bodies. An E2 analog was identified in the hemolymph of day 3 wandering silkworms using high-performance liquid chromatography. Estradiol titers from fifth late-instar larvae to pupal stage were determined by enzyme-linked immunosorbent assay. The results suggested that silkworms synthesized a vertebrate E2 analog. This study found that E2 promoted the synthesis of BmVg, a female typical protein in silkworms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Diapause was induced in polyvoltine silkworm B. mori eggs and the molecular mechanism involved in diapause was investigated using a genome wide microarray. In diapause eggs, 638 and 675 genes were upregulated, while, in non-diapause eggs 1136 and 595 genes were upregulated at 18 h and 30 h, respectively after oviposition. Real-time qPCR analysis confirmed the expression of 20 genes, and the relative expression levels of the Aquaporin gene was highest among the 20 genes, followed by Sorbitol dehydrogenase-2 and Cytochrome b5 in diapause eggs, while, Kruppel homolog, Period and Relish were higher in non-diapause eggs. The upregulation of SDH-2 and cytochrome b5 indicates increased metabolic rate in diapause-destined embryos prior to the onset of diapause within 36 h as a preparatory phase. This study provides an insight into the early molecular events for the induction and maintenance of diapause in B. mori., (© 2013 Elsevier Inc. All rights reserved.)

Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Silkworm (Bombyx mori) is an economically important insect. However, non-cocoon caused by chemical insecticide poisoning has largely hindered the development of sericulture. To explore the roles of detoxification enzymes in B. mori after insecticide poisoning, we monitored the activity changes of cytochrome P450 monooxygenase, glutathione-S-transferase, and carboxylesterase in B. mori midgut and fatbody after phoxim feeding. At the same time, the expression levels of detoxification enzyme-related genes were also determined by real-time quantitative PCR. Compare to the control levels, the activity of P450 in the midgut and fatbody was increased to 1.72 and 6.72 folds; the activity of GST was no change in midgut, and in fatbody increased to 1.11 folds; the activity of carboxylesterase in the midgut was decreased to 0.69 folds, and in fatbody increased to 1.13 folds. Correspondingly, the expression levels of detoxifying enzyme genes CYP6ae22, CYP9a21, GSTo1 and Bmcce were increased to 15.99, 3.32, 1.86 and 2.30 folds in the midgut and to 3.58, 1.84, 2.14 and 4.21 folds in the fatbody after phoxim treatment. These results demonstrated the important roles of detoxification enzymes in phoxim metabolism. In addition, the detected activities of such enzymes were generally lower than those in cotton bollworms (Helicoverpa armigera), which may contribute to the high susceptibility of B. mori to insecticides. Our findings laid the foundation for further investigations of the molecular mechanisms of organophosphorus pesticide metabolism in B. mori., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In insects, especially phytophagous insects, feeding behavior occurs at a regular frequency. Although a number of physiological studies have revealed various causal factors leading to feeding behavior in insects, little has been demonstrated regarding the regulatory mechanisms underlying insect feeding behavior. To confirm the presence of an endocrinological regulatory mechanism in feeding behavior, we tested the effects of several biologically active peptides on silkworm, Bombyx mori larvae feeding behaviors. To evaluate the effects of the biologically active peptides, we measured the period of latency to the first bite following sample injection into starved Bombyx larvae. Of the chemically synthesized peptides tested, myosuppressin exhibited a prolonged latency, indicating that myosuppressin is a possible inhibitory peptide in Bombyx larvae. In contrast, injections of tachykinin and short neuropeptide F, which are members of the structurally related RF-amide peptide family, had a shorter latency period, indicating that these two peptides are possible stimulatory peptides. In addition, the present study suggests that this bioassay will be advantageous for screening for peptides that regulate insect feeding behavior., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal. The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish., ((c) 2010 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

Staphylococcus aureus is a pathogenic bacterium that causes abscesses, pneumonia, endocarditis, and food poisoning. S. aureus is also one of the resident flora of the endotherm and colonizes the host by skillfully evading its defense mechanism. Identification of attenuated mutants of S. aureus in an animal infection model is useful for investigating its adaptability and pathogenesis. This chapter describes a staphylococcal two-component SA0614-SA0615 system, which was identified using a silkworm larvae infection model.

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.

Insect growth, development, and molting depend upon a critical titer of the principal molting hormone of arthropods, 20-hydroxyecdysone (20E). Although the structure of 20E as a polyhydroxylated steroid was determined more than five decades ago, the exact steps in its biosynthesis have eluded identification. Over the past several years, the use of the fly database and the techniques and paradigms of biochemistry, analytical chemistry, and molecular genetics have allowed the cloning and sequencing of four genes in the Halloween gene family of Drosophila melanogaster, all of them encoding cytochrome P450 (CYP) enzymes, each of which mediates one of the four terminal hydroxylation steps in 20E biosynthesis. Further, the sequence of these hydroxylations has been determined, and developmental alterations in the expression of each of these genes have been quantified during both embryonic and postembryonic life.

Isolated brain-retrocerebral neurohemal complex of the silkworm of Bombyx mori was stimulated electrically and the released bombyxin (an insulin-like neuropeptide) was measured using time-resolved fluoroimmunoassay. The amount of bombyxin release depended on the number of stimulus pulses delivered to the axonal tract of the bombyxin-producing (BP) neurosecretory cells, and 17 fg of bombyxin per pulse was released from a cell. The titer of bombyxin in the hemolymph of bombyxin-II injected pupae decreased exponentially, the half-life being 170 min. To relate firing activity of a population of BP cells to the hormone titer in the hemolymph, bombyxin titer and its change in the hemolymph were calculated numerically. We assumed that the amount of bombyxin release was proportional to the firing rate of BP cells and the released bombyxin was inactivated with the same time course of injected bombyxin. Our calculations suggested that the hemolymph bombyxin titer may fluctuate dynamically and the mean titer is 380 pg/ml, a level which is close to the actually determined bombyxin titer at middle stages of pupal-adult development.

Mode of action by which entomopoxvirus (EPV) spindles enhance nucleopolyhedrovirus (NPV) infection remains unclear. Spindles of Anomala cuprea entomopoxvirus (AcEPV), a coleoptran EPV, are known to enhance Bombyx mori NPV (BmNPV) infection in silkworm larvae. AcEPV spindles were orally administered to silkworm larvae with or without BmNPV polyhedra, and the peritrophic membranes (PMs) were observed using a binocular microscope. Soon after the larvae's access to spindles with or without the polyhedra had been terminated, some PMs disappeared wholly and some were observed in partial form. Some of the partial PMs observed were very fragile. The disintegration of the PM due to spindles also was observed by the histological sectioning of the midgut. However, a day after the larvae had terminated their access to the spindles, the PM regenerated partially or wholly. In contrast, the administration of AcEPV spheroids caused neither the disintegration of PMs nor the enhancement of BmNPV infection in silkworm larvae. These findings strongly suggest that the enhancement of NPV infection occurs due to that a greater number of NPV virions reaching the microvilli of midgut susceptible to NPV, since spindles lead to the disintegration of the PM as a barrier against NPV virions.

When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar., (Copyright 2002 Elsevier Science (USA))

Silkworm larvae, Bombyx mori, were examined as an animal model of human infection with pathogenic bacteria. When 3 x 10(7) cells of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa, or Vibrio cholerae were injected into the blood of fifth instar silkworm larvae, over 90% of the larvae died within 2 days, whereas over 90% survived for 5 days after injection of the same amount of Escherichia coli. Growth of S. aureus was observed in larvae blood and tissues. Immunostaining analysis revealed that S. aureus proliferated at the surface of the midgut. Infection of silkworm larvae by methicillin-sensitive S. aureus was cured by ampicillin, oxacillin, and vancomycin, whereas infection by methicillin-resistant S. aureus was not cured by ampicillin or oxacillin, although vancomycin was effective. Disinfectants were not effective because of toxicity against the larvae. Thus, silkworm larvae are useful for evaluating antibiotics for pathogenic bacterial infection in humans., (Copyright 2002 Academic Press.)

DNA synthesis in the prothoracic gland cells of the silkworm Bombyx mori was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the last larval instar were examined. During the first 3 days of the last larval instar, no DNA-synthesizing cells were detected. On day 4, the number of DNA-synthesizing cells increased and peaked on day 7. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, the number of DNA-synthesizing cells dramatically increased 2 days after 20-hydroxyecdysone treatment and reached a high level on day 5. The mechanism by which 20-hydroxyecdysone treatment induces DNA synthesis of prothoracic gland cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. We conclude that 20-hydroxyecdysone may exert its growth-promoting action indirectly.

Although estradiol-17beta (E2) is known to be a vertebrate sex steroid, its presence in insects, particularly in Bombyx mori, has recently created considerable sensation regarding its significance in insect life. The specificity of estradiol action on the posterior silk gland (PSG) of B. mori has been previously assessed by the use of ICI-182780 as a specific receptor antagonist for estradiol. The present study provides evidence for systematic changes in the titer of estradiol-17beta in B. mori and its specific binding sites in the posterior silk gland according to the physiological age of fifth instar larvae. The changes in the estradiol level in the PSG after E2 treatment have also been determined to assess whether the administered estradiol has been taken up by the silk gland cells and whether the effect of exogenously administered E2 on the PSG is direct. The correlation in the profile of E2 binding sites with the endogenous estradiol level of PSG on different days of fifth instar larvae suggests that the level of binding sites in the PSG is regulated by the estradiol titer and that E2 may have some biological function in B. mori. Scatchard analysis of E2 binding sites in the PSG on 9th day of fifth instar larvae reveals that it consists of only one type of binding component. The dissociation constant (Kd) and maximum binding capacity (Bmax) of this putative E2 receptor have been found to be 8.98 +/- 0.744 nM and 55.8 +/- 5.53 fmol/mg cytosolic protein, respectively. The study thus indicates an involvement of the putative receptor in E2-mediated action on the silk gland of B. mori and paves a pathway for further evaluation of the physiological relevance of the presence of vertebrate steroids in insects.

A prothoracicostatic peptide (PTSP), purified from the brains of Bombyx mori, was found to inhibit ecdysteroidogenesis in the prothoracic glands (PGs) of this insect. This peptide was active at inhibiting ecdysteroidogenesis in the PGs at concentrations higher than 23 nM and glands incubated in 230 nM PTSP in vitro exhibited maximum inhibition of ecdysteroid production. By incubating PGs in vitro at different incubation periods it was observed that the first statistically significant inhibitory effect occurred after 30 min incubation in the presence of PTSP. Transferral of PGs from a medium with PTSP to a medium without PTSP resulted in the resumption of ecdysteroid production. Statistically significant inhibition of ecdysteroid production by PTSP was observed only in day 6 and in day 3 PGs of the 5th instar. The extracts of day 6 glands incubated in the presence of PTSP did not contain elevated amounts of ecdysteroid relative to controls after the incubations, indicating that PTSP does not inhibit the secretion, rather the synthesis, of ecdysteroid in the PGs. The presence of PTSP completely blocked the increased ecdysteroid production via L-type Ca(2+) channel activation by S(-)*Bay K 8644. There was no inhibition of ecdysteroid production by PTSP with glands incubated in Ca(2+)-free medium. The combined results suggest that PTSP regulates ecdysteroid synthesis only during specific stages of the 5th instar through a mechanism that likely involves the blocking of Ca(2+) influx through voltage-sensitive Ca(2+) channels in the PG cells of B. mori., (Copyright 2001 Academic Press.)

This study focuses on the effect of octopamine (OA) on metamorphosis of the silkworm Bombyx mori and the red flour beetle Tribolium freemani Hinton. Titers of OA and juvenile-hormone esterase (JHE) were measured at various larval and pupal stadia of both insects. Effects of OA, OA agonists, and antagonists on metamorphosis and JHE activity were also examined. At day 2, peaks of OA and JHE activity were observed in third instars, and at day 3, a sharp peak of OA was observed, followed by a large peak of JHE activity at day 4 in last instars of B. mori. However, no peaks of OA and JHE activity were observed in fourth instars. A high titer of OA appeared at days 2-4, followed by a peak of JHE activity at day 7 and the second OA peak at day 9 after the start of assay of T. freemani. At pupation, a small peak of OA and the highest activity of JHE were observed. The effects of OA on JHE activity were examined in vitro, because the relationship could be responsible for triggering pupation in B. mori and T. freemani larvae. Exogeneous OA (0.1-10 mM) stimulated the JHE activity of final instars (day 2) of B. mori in vitro. Similarly, the presence of OA (10 mM) activated the JHE activity of newly ecdysed T. freemani pupae in vitro. OA antagonists chlorpromazine and gramine delayed the start of spinning and reduced the JHE activity of B. mori, when applied in diet at 10-100 ppm. Some OA agonists stimulated the pupation and JHE activity of T. freemani larvae reared under crowded conditions, when topically applied. Thus, OA may contribute to activation of the events preparatory to a pupal molt, i.e., the secretion of OA increases JHE activity followed by stimulation of pupation., (Copyright 1999 Academic Press.)

We investigated the effects of two ecdysteroids, ecdysone (E) and 20-hydroxyecdysone (20E), on silkworm larval development. Silkworm larvae, Bombyx mori, were fed an artificial diet supplemented with 20E during the fourth instar to promote premature molting. At the onset of the fifth instar, these precocious fifth-instar larvae were fed diets supplemented with either E or 20E to determine the effects of the two ecdysteroids on the morphology of midgut epithelial cells. Regeneration of midgut epithelial cells normally occurs only during the molting period. However, in larvae fed E, complete replacement of midgut epithelial cells was observed 24 h before the larvae entered apolysis. In larvae fed 20E, the morphology of midgut epithelial cells was disrupted, leading to death of the larvae during the fifth instar. We also observed similar differences in the effects of the two ecdysteroids in an in vitro experiment. These results suggest that E has a specific effect on the morphological change of midgut epithelial cells in precocious fifth-instar larvae that is distinct from 20E., (Copyright 1999 Academic Press.)

Temporal changes in DNA synthesis were examined in the corpora allata of the silkworm Bombyx mori during the penultimate larval stadium. 5'-Bromo-2'-deoxyuridine (BrdU) labeling revealed that allatal DNA synthesis rose early in the stadium, peaking at about 2% of corpus allatum (CA) cells 24 hr after ecdysis. This corresponded to a rising phase of the ecdysteroid titer reported previously. DNA synthesis declined to a nearly undetectable level 1 day later. In larvae fed on leaves coated with 20-hydroxyecdysone (20E), the number of BrdU-labeled cells increased continuously after the normal peak and reached about 8% of CA cells at the end of the stadium. However, total count of CA cells from whole-mount monolayers and colchicine treatment indicated that, regardless of DNA content, CA cells did not divide. To determine if the rise in 20E was responsible for the onset of allatal DNA synthesis, we have established an organ culture system to support de novo DNA synthesis in vitro. The number of BrdU-labeled cells increased with increasing 20E concentration between 0.1 and 0.5 microg ml-1 in the medium, corresponding well to the rising 20E titer in the hemolymph. The level of DNA synthesis in culture was further elevated when 20% hemolymph was added together with 20E, suggesting that other direct stimulatory factors for allatal DNA synthesis may also exist in the hemolymph.

Effects of fenoxycarb on the secretory activity of the prothoracic glands (PGs) of the silkworm, Bombyx mori, were investigated. Fenoxycarb inhibited the secretory activity of PGs in vitro throughout the fifth instar. Incubation of PGs in the presence of 1 microgram of fenoxycarb resulted in a approximately 50% inhibition of their secretory activity. This inhibition of fenoxycarb was dose dependent. Stimulation of the PGs by recombinant prothoracicotropic hormone (rPTTH) reached a high activation ratio of 15.9. Simultaneous presence of fenoxycarb and 1 ng rPTTH significantly decreased the stimulatory effect of rPTTH on the PGs, and the activation ratio never exceeded 3.65. Early in the fifth instar, fenoxycarb strongly inhibited the secretory activity of the PGs and induced a developmental arrest as well. After the onset of pupal commitment, its action toward the PG secretory activity was inhibitory. However, fenoxycarb treatment was not able to induce a developmental arrest in intact animals after the pupal commitment although it could do so in head-ligated animals. On the contrary, application of fenoxycarb after the wandering behavior resulted in a significantly increased secretory activity by the PGs of both intact and head-ligated larvae.

Since estradiol has been detected in Bombyx ovaries effects of estradiol and other steroids on the growth and maturation of the silkworm ovary, rate of oviposition, and embryonic development were examined as a part of a study aimed at the clarification of physiological significance of estradiol in insects. These steroids were injected at various doses into the whole pupae and into the isolated pupal abdomens. No significant effect by the injections was observed on the ovarian development, as judged by increase in protein content or wet weight of ovaries and pattern of protein constituents including vitellin. However, rate of oviposition was considerably affected by the injection of estradiol at high doses. No clear effect was observed on embryonic development or determination of diapause by the injection of estradiol into the pupae. Effects of injection of anti-estrogen, nafoxidine, into the isolated abdomens and whole pupae were also examined. No effects were observed on ovarian development by injection. The relationship between physiological significance of the vertebrate steroids and metabolic activity of the ovary is discussed.