Instar Larvae Of Silkworm Research Articles

Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection

Sonoporation (ultrasound treatment) provides a new and attractive nonviral way of in vivo gene transfer. To access the applicability of this method to the silkworm, Bombyx mori, we have compared the efficiencies of gene transfer by means of lipofection (using an appropriate agent, PDD111), sonoporation (ditto, FluoroGene), and lipofection followed by sonoporation. By these methods, a luciferase expression plasmid was found to be markedly transferred into the haemocoel of newly ecdysed fifth instar silkworm larvae, and also into other tissues although with lower rates compared with the haemocoel. In terms of luciferase activity, the efficiencies of transgene by lipofection plus sonoporation were approximately 6 (hemocytes), 20 (silk glands), 8 (mid-gut), 38 (fat body), 10 (Malpighian tubules), 33 (ovaries), and 16 (testes) times as high as those by lipofection or sonoporation alone. These results demonstrated that the present method is useful to introduce the exogenous DNA into insect organs in vivo.

Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system

To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.

Analysis of ESTs and gene expression patterns of the poste-rior silkgland in the fifth instar larvae of silkworm, Bom-byx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.

A constitutive super-enhancer: homologous region 3 of Bombyx mori nucleopolyhedrovirus

Transient expression assays were carried out to assess the transcriptional enhancement from the immediate-early gene ( ie-1 ) promoters by cis -linked Bombyx mori nucleopolyhedrovirus (BmNPV) homologous region-3 ( hr3 ). The ie-1 promoters were derived from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and BmNPV. Hr3 was placed downstream of the luciferase ( luc ) gene, the resulting plasmids were used to transfect the Spodoptera frugiperda (Sf-21), Bombyx mori (Bm-5, Bm-N) cell lines and 5th instar silkworm larvae mediated by lipofectin. The results revealed that hr3 could stimulate the transcription of homologous BmNPV ie-1 promoter 1970, 2421, 683, and 1059 folds, and the heterologous AcMNPV ie-1 promoter 6462, 4046, 6980, and 605 folds, respectively. These results implied that hr3 was a super enhancer which could function both in vitro and in vivo. The 30-bp imperfect palindrome in hr3 was proved to be the essential structure for enhancing function of hr3 . The utilizing of this promoter–enhancer combination in the baculovirus expression vector system (BEVS) was also carried out to further confirm the function of hr3 enhancer.

A constitutive super-enhancer: homologous region 3 of Bombyx mori nucleopolyhedrovirus

Transient expression assays were carried out to assess the transcriptional enhancement from the immediate-early gene ( ie-1) promoters by cis-linked Bombyx mori nucleopolyhedrovirus (BmNPV) homologous region-3 ( hr3). The ie-1 promoters were derived from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and BmNPV. Hr3 was placed downstream of the luciferase ( luc) gene, the resulting plasmids were used to transfect the Spodoptera frugiperda (Sf-21), Bombyx mori (Bm-5, Bm-N) cell lines and 5th instar silkworm larvae mediated by lipofectin. The results revealed that hr3 could stimulate the transcription of homologous BmNPV ie-1 promoter 1970, 2421, 683, and 1059 folds, and the heterologous AcMNPV ie-1 promoter 6462, 4046, 6980, and 605 folds, respectively. These results implied that hr3 was a super enhancer which could function both in vitro and in vivo. The 30-bp imperfect palindrome in hr3 was proved to be the essential structure for enhancing function of hr3. The utilizing of this promoter–enhancer combination in the baculovirus expression vector system (BEVS) was also carried out to further confirm the function of hr3 enhancer.

Detection of Proteins with a High Affinity for Imidazole Insect Growth Regulator, KK-42

For explanation on mode of action of KK-42, an imidazole insect growth regulator, an amine analog of KK-42 (1-(3-aminopropyl)-5-[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole, ADHI) has been designed and prepared as a ligand to detect proteins with high affinity in pharate first larvae of wild silk moth associated with artificial hatching. The mode of action of KK-42 for induction of precocious metamorphosis is speculated to be an inhibition of ecdysteroid biosynthesis at the prothoracic gland (PG). Since the PG in 5th instar larvae of silkworm on day 7 has strong biosynthetic activity and is sensitive to KK-42, a supernatant of a homogenate of the PG was applied to ADHI-Sepharose. After optimization of the condition of affinity chromatography, a single protein was eluted with free KK-42. This protein did not bind to the affinity gel when incubated with KK-42 before the application.

Immune stimulation in the silkworm, Bombyx mori L., by CpG oligodeoxynucleotides.

Synthetic ODNs containing unmethylated CpG dinucleotides are known to stimulate immune responses in vertebrates, but so far the effect has not been studied in insects. In this report, we describe an induction of immune response following injection of oligodeoxynucleotides (ODNs) into the insect hemocoel. The fifth instar silkworm (Bombyx mori L.) larvae were injected with several synthetic ODNs containing variable number of unmethylated CpG motifs, heat-denatured genomic DNA of B. mori itself, or intact genomic DNA to observe a new induction pattern in the insect immune mechanism. When the induction of immune response was examined based on the expression rates of genes for antibacterial peptides such as attacin and cecropin, we could confirm that it was triggered upon injection of ODNs. The expression was, however, neither dependent on numbers of CpG motifs nor methylation of CpGs in ODNs. Furthermore, it was confirmed that the presence of CpG in ODN was not involved in the induction pattern of insect immunity caused by ODNs, although it has been reported that vertebrates respond in a specific manner against invading ODNs containing CpG dinucleotides. In addition, insect immunity was not stimulated by injection of intact DNA from host. In contrast, the injection of denatured genomic DNA provoked the host immune reaction. Taken together, our data suggest that foreignness of ODNs or DNA might be a key factor in the induction of insect immunity.

Preparation of protein samples for gel electrophoresis by sequential extraction

Since preparation and solubilization of protein samples are crucial factors in proteome research, the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm,Bombyx mori. Two kinds of protein samples were obtained from the body wall using the method. Between the two types of samples only about 15% proteins were identical; the majority were different, indicating that more species of proteins could be obtained with the sequential extraction method; which will be useful for preparation of protein samples for proteome study.

Silkworm larvae as an animal model of bacterial infection pathogenic to humans

Silkworm larvae, Bombyx mori, were examined as an animal model of human infection with pathogenic bacteria. When 3×107 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.

Foreign insect hormones stimulating the transcription of the ie-1 promoter of Bombyx mori nuclear polyhedrosis virus in vivo and in vitro.

Via a transient expression assay system, an experimental study was undertaken to characterize the effects of insect ecdysone and juvenile hormone analogue on the transient expression of the luciferase gene under the control of the immediate-early gene (ie-1) promoter of Bombyx mori nuclear polyhedrosis virus. The results demonstrated that the transcriptional activity of the ie-1 promoter was increased to a certain extent by different insect hormone treatments in uninfected insect cells or fifth instar silkworm larvae transfected with a plasmid containing a luciferase gene driven by the ie-1 promoter. By ecdysone treatment alone, an increase of 5-7 fold was reached in Bm-N, or Bm-5 cells, or in the early developmental stage of fifth instar larvae. By treatment with juvenile hormone analogue alone, about 2-fold, in Bm-N, Bm-5, and Sf-21 cells, or about 5-fold increase in the middle developmental stage of larvae was given, respectively. By co-treatment with ecdysone and juvenile hormone analogue, the incease was given between that of ecdysone and juvenile hormone analogue treatment alone. In addition, the synergistic effects of foreign/endogenous hormones on the activity of ie-1 promoter are discussed.

Insecticidal compounds from Kalanchoe daigremontiana × tubiflora

Methyl daigremonate, an insecticidal bufadienolide, was isolated from the leaves of Kalanchoe daigremontiana× tubiflora (Crassulaceae) along with four known bufadienolides. Its structure was established by spectroscopic analysis, and insecticidal activities were assessed against the third instar larvae of silkworm ( Bombyx mori). The results suggest that the orthoester and α-pyrone moieties played an important role in the activity.

Effects of jinlu, an anti-juvenile hormone on the growth, ultra-structure of the corpora allata and prothoracic gland of silkworm,Bombyx mori L

The 4th instar larvae of the silkworm,Bombyx mori L, when treated with anti-juvenile hormone (Jinlu) had its larval period extended by 2 days and the total larval period shortened by about 4 days. The conversion ratio of tetramolters into trimolters was 100%. But anti-juvenile hormone administration to the 5th instar larvae lengthened the silkworm age by one day. When anti-juvenile hormone was administered, we could find many neurosecretory granules of the brain transferred to the cells of the corpora allata, but there was little endoplasmic reticulum. In the prothoracic gland, the micropile edge was clear and there were large nucleoli, mitochondria and endoplasmic reticulum. This study was carried out to show that anti-JH compound inhibits the secretion of Juvenile hormone in silkwormBombyx mori L. The investigation revealed that the antijuvenile hormone inhibited the secretion of corpora allata and initiated the activity of the prothoracic gland.

Identification of Highly Active Genes from the Posterior Silk Gland of the Oak Silkworm, Antheraea yamamai, Through Expressed Sequence Tag (EST) Strategy

Gene discovery from highly specialized tissues is important for understanding of development and tissue-dependent gene regulation in organism. To identify highly active genes from the posterior silk gland of the oak silkworm, Antheraea yamamai, which is specialized for the production of silk proteins, we constructed an oligo (dT) primed directional cDNA library from the posterior silk gland of the final star larvae. One hundred and three expressed sequence tags (ESTs) were generated by single-run partial sequencing from the primary cDNA library. Their sequence analysis indicated that 59 clones exhibited a significant homology to known genes, and 6 clones to ESTs of unknown functions, and 33 clones were novel transcripts. In the expression profile, the most abundant gene was found to be fibroin (25.2%), followed by TAXREB107 (5.8%) and ribosomal protein S9 (2.9%). Northern blot analyses confirmed that fibroin and TAXREB107 genes are actively expressed in the posterior silk gland of the final instar larvae of the oak silk worm. This study demonstrated that the expression profile of the silk gland ESTs could be a useful tool for isolating the highly active genes in insect tissues.

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24 000, 48 000 and 23 000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization–time of flight/mass spectrometry (MALDI–TOF/MS), the respective molecular masses were determined to be 22 641, 22 908 and 22 737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IX γ. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.

Evidence for Growth of Strains of the Plant Epiphytic BacteriumErwinia herbicolaand Transconjugation among the Bacterial Strains in Guts of the SilkwormBombyx mori

Growth of plant epiphytic bacteriaErwinia herbicolaandPseudomonas syringaein guts of the silkworm,Bombyx mori,was studied. Fifth instar silkworm larvae were fed artificial diets supplemented with these bacteria for 6 to 12 h followed by uncontaminated diets. At 1, 3, and 6 days after feeding, bacteria were isolated from insect guts and feces. A much larger population ofE. herbicolawas detected in the samples collected 3 and 6 days after the inoculation than in samples collected after 1 day, indicating that these bacteria grew in the insect gut, whileP. syringaewas unable to survive. Transconjugation betweenE. herbicolastrains in the insect gut was also examined. First, either a donor or a recipient strain was fed to the insects in artificial diets containing the bacteria during 12 h, and then pairing strains were fed during 12 h after starvation for 12 h. The conjugative plasmid pBPW1::Tn7was transferred into recipient cells at very high frequencies (10−1/recipient after 3 days and 10−3after 6 days) in insect guts. Indigenous plasmids ofE. herbicolamobilized RSF1010 plasmid into recipient cells at frequencies of 10−4in insect guts. These transconjugants were detected in the feces of the insects. Thus, plasmid-mediated gene transfer among the epiphytic bacteria in insect guts was demonstrated. The results obtained suggest thatin insectagene transfer may play an important role in the evolution of plant epiphytic bacteria.

Sperm-mediated gene transfer in the silkworm Bombyx mori.

Plasmid DNA containing the CAT reporter gene was injected into the testis of V instar silkworm larvae. The persistence, expression, and transmission of the injected DNA were monitored in the injected individuals till eclosion as well as in the progeny. The DNA injected into the testis persisted extrachromosomally during the entire period of metamorphosis and was also transferred into the egg via sperm during fertilization. Injected plasmids were rescued from the moths that emerged from the injected larvae and also from the eggs laid by the moths that copulated with injected males. Positive signals for CAT assay in the experimental samples suggested that the injected DNA was internalized in the testicular cells and sperm. The persistence, expression, and transmission of the DNA injected into the testes indicate that sperm-mediated gene transfer is possible in the silkworm, Bombyx mori.

Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus.

We have examined the efficiency of coexpression of two heterologous genes from a recombinant Bombyx mori nuclear polyhedrosis virus for the production of antibodies in silkworm larvae. The cDNAs encoding the light and the heavy chains of a murine immunoglobulin, directed against lipoprotein I of Pseudomonas aeruginosa, were brought under the control of two separate copies of the viral polyhedrin promotor. Infection of silkworm cells with the recombinant baculovirus yielded a maximum of 6.4 micrograms/ml IgG2A in the culture supernatant 72 hours post infection, while 800 micrograms/ml IgG2A was found in the hemolymph of infected fifth instar silkworm larvae seven days after infection with the same construct. The recombinant antibody exhibited a similar antigen specificity and avidity to that of the monoclonal antibody derived from ascites fluid.

Structure and Insecticidal Activity of New Indole Alkaloids, Okaramines A and B, fromPenicillium simplicissimumAK-40

Penicillium simplicissimum AK-40, which was isolated from a soil sample, produced novel insecticidal compounds when cultured with okara (the insoluble residue of whole soybean). The metabolites, named okaramine A (1) and B(2), were crystalline products with molecular formulas C32H32N4O3 and C33H34N4O5, respectively. The structure of 1 was deduced from the structure of acetylokaramine A that was established by single crystal X-ray diffraction. The structure of 2 was determined from the data of UV, IR, NMR (1H and 13C) and mass spectrometry. 1 and 2 showed insecticidal activity against the 3rd instar larvae of silkworm at doses of 3 μg/g of diet and 0.1 μg/g of diet, respectively.

Structure and insecticidal activity of new indole alkaloids, okaramines A and B, from Penicillium simplicissimum AK-40.

Penidllium simplidssimum AK-40, which was isolated from a soil sample, produced novel insecticidal compounds when cultured with okara (the insoluble residue of whole soybean). The metabolites, named okaramine A (1) and B(2), were crystalline products with molecular formulas C32H32N4O3 and C33H34N4O5, respectively. The structure of 1 was deduced from the structure of acetylokaramine A that was established by single crystal X-ray diffraction. The structure of 2 was determined from the data of UV, IR, NMR (1H and 13C) and mass spectrometry. 1 and 2 showed insecticidal activity against the 3rd instar larvae of silkworm at doses of 3 μ/g of diet and 0.1 μg/g of diet, respectively.

Transovarial transmission of two microsporidia in the silkworm, Bombyx mori, and disease occurrence in the progeny population

Peroral inoculation of Nosema bombycis and Nosema sp. M11 spores to 5th instar silkworm larvae was the most suitable method for obtaining infected adults for transovarial transmission studies. Mating tests with infected male and uninfected female adults indicated that N. bombycis and Nosema sp. M11 were not venereally transmitted. However, N. bombycis was transmitted transovarially to the progeny eggs at a frequency of 100%, whereas Nosema sp. M11 was transmitted at only 1.2 ± 0.41%. In larval progeny of the F 1 generation that were infected transovarially with N. bombycis, an acute lethal infection developed and most larvae died by the end of the 3rd stadium. Transovarial transmission of Nosema sp. M11, however, produced a chronic lethal infection and larval death did not occur until the end of the 5th stadium, when mortality increased up to 1.0% before eclosion.