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Pigment pattern formation in the quail mutant of the silkworm, Bombyx mori: parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes.

The larval pigment pattern in the silkworm, Bombyx mori, is formed by melanin, ommochromes and pteridines. During development all these pigments are synthesized autonomously, and possibly also with mutual interaction between them, to yield unique pigment patterns. In order to find the key trigger for such pigment pattern formation, developmental changes in pteridine biosynthesis were studied using the quail mutant (q/q), which has darker larval marks formed by melanin and an abundance of ommochromes in the integument. In the current study, emphasis has been placed on the analysis of GTP-cyclohydrolase I (GTP-CH I), which is a key enzyme for the biosynthesis of pteridines, during the development of the silkworm. Results of Northern blotting showed that in the quail mutant strong signals of GTP-CH I mRNA appeared around each period of ecdysis, while no such signals appeared in the background strain (+q/q) used. Also, both GTP-CH I activities and pteridine content were higher in the quail mutant compared with the background strain. These results strongly suggest that pteridine biosynthesis is closely linked to the formation of melanin and ommochromes. It is also suggested here that in the silkworm a recessive gene (q) may be involved in the regulation of its pigment pattern formation.

Alcohol Oxidoreductases↗

Bactericidal activity of the normal, cell-free hemolymph of silkworms (Bombyx mori).

Cell-free hemolymph from silkworm (Bombyx mori) larvae can kill Escherichia coli B/SM. The bactericidal principle can be resolved into at least two factors. One is a lysozyme-like enzyme that can be absorbed on crab shell chitin and on bentonite, and the other (cofactor) is an anionic factor that is of low molecular weight, can pass through the chitin column and a carboxymethyl-cellulose column, and can be eluted from a diethylaminoethyl-cellulose column at mu = 0.15 and pH 7.5. Egg-white lysozyme cannot replace the silkworm lysozyme-like enzyme for restoring the bactericidal activity when it is mixed with the cofactor, although it can enhance the bactericidal activity of the mixture of silkworm enzyme and cofactor. Mg2+ and Ca2+ can inhibit the bactericidal activity.

Animals↗

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.

Animals↗

Photosensitivity in the circadian hatching rhythm of the carotenoid-depleted silkworm, Bombyx mori.

Silkworms (Bombyx mori) were reared on a carotenoid-deprived artificial diet, and the carotenoid-depleted eggs of the next generation were incubated so that we could observe the effect of the depletion on the circadian rhythm of hatching. The phototactic response curves of newly hatched larvae showed that the visual photosensitivity in ocelli of larvae from the carotenoid-depleted eggs was at least 4 log units lower than that of a carotenoid-rich control group. However, the phase-shift experiment revealed that carotenoid depletion did not reduce the photosensitivity in the hatching rhythm. When the hatching rhythm was generated by exposure to a single light pulse in constant darkness, the first peak in the rhythm of the carotenoid-depleted silkworms occurred significantly earlier than that of the carotenoid-rich group, but the following second peaks of both groups were found at the same time. These results suggest that for the silkworm, carotenoid is not involved in photoreception for the hatching rhythm, but is involved in the timing of hatching.

Animals↗

Transposable elements in commercially useful insects: I. Southern hybridization study of silkworms and honeybees using Drosophila probes.

As a first step in surveying transposable elements in silkworms and honeybees, hybridization analyses were carried out using 16 known families of Drosophila transposable elements as probes. jockey and G were the only transposable elements that hybridized with genomic DNA of either honeybees or silkworms under the conditions of this study. jockey hybridized with genomic DNA of both European honeybees (Apis mellifera) and silkworms (Bombyx mori and Antheraea yamamai) and showed significant bands in Southern blots. Banding patterns were highly polymorphic. jockey did not, however, hybridize with any strains of the Asian honeybee (A. cerana). G elements showed a faint signal with the Asian honeybee, but not with any other insects tested. The results suggest that, even though it has some limitations, this approach can be used in practice as a first preliminary step in surveys for the presence of transposable elements in organisms which do not have good genetic information.

Animals↗

Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system.

In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.

Animals↗

Establishment of a large-scale purification procedure for purified recombinant bovine interferon-tau produced by a silkworm-baculovirus gene expression system.

We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.

Animals↗

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx 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.

Animals↗

[Comparison of early embryogenesis between silkworm and Drosophila from both morphology and gene content level].

The diversity of insects has been the focus of many investigations. Research in the early embryonic developmental mechanism of silkworm will promote understanding in early embryonic developmental mechanism of other insects. This paper reviews the differences in early embryonic development of silkworm and Drosophila from both morphology and gene content level. The results not only provide evidences for the study of early embryonic development mechanism in silkworm, but also give clues for the study of insects diversity.

Animals↗

Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori.

beta-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing beta-D-glucose residues from the end of various oligosaccharides with the release of beta-D-glucose. CelB gene, encoding the thermostable beta-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express beta-glucosidase in silkworm. The recombinant beta-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed beta-glucosidase was obtained at pH 5.0 and about 105 degrees C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant beta-glucosidase were similar to the native counterpart. The expressed beta-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable beta-glucosidase using this system.

Animals↗

[Expression of human GDNF in methyltrophic yeast Pichia pastoris and silkworm larvae].

The cDNA encoding glial cell derived neurotrophic factor (GDNF) was cloned into the Pichia expression vector pPIC9K and then transformed into his4 mutant yeast GS115 by electroporation. Multicopy transformants were screened by various G418 concentrations and induced by methanol. The human GDNF gene was cloned into the baculovirus transfer vector pBacPAK8. The recombinant transfer vector pBacPAK-GDNF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. SDS-PAGE and Western blot confirmed that GDNF was expressed in Pichia culture medium and silkworm larvae hemolymph. The GDNF protein expressed in Pichia and silkworm larvae could significantly promote the survival and neurite outgrowth of dopaminergic neurons.

Animals↗

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori.

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.

Animals↗

Enhancing effects of silkworm expressed recombinant human macrophage colony-stimulating factor on hematopoietic recovery of irradiation-injured mice.

OBJECTIVE: To study the hematopoietic enhancing effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) expressed in silkworm. METHOD: Balb/c mice were irradiated with sublethal dose of 60 Co gamma-rays and then administered intraperitoneally with silkworm expressed rhM-CSF (1000 U per individual for 7 days) in treatment group or with normal saline (for 7 days as well) in control group. The hematopoietic recovery of irradiation mice was observed by comparing peripheral white blood cell (WBC) counts, differential counts of WBC and bone marrow hematopoietic progenitor cell colony forming assay in soft agar at different time after irradiation. RESULTS: The total WBC counts (x 10(9)/L) of treatment group at day 15 and 20 after irradiation(3.42 +/- 1.20, 5.56 +/- 2.50, respectively) were significantly higher than those of control group (2.03 +/- 0.90, 3.72 +/- 2.30; both P < 0.05). On days 10, 15, 20, 25 and 30 after irradiation, the monocyte counts (x 10(9)/L) of treatment group (0.08 +/- 0.06, 0.16 +/- 0.10, 0.48 +/- 0.35, 0.47 +/- 0.21 and 0.33 +/- 0.17, respectively) were all significantly higher than those of control group (0.025 +/- 0.016, 0.05 +/- 0.04, 0.23 +/- 0.16, 0.33 +/- 0.19 and 0.17 +/- 0.13; all P < 0.05). On days 15, 20 and 25, the granulocyte count (X 10(9)/L) of treatment group (1.03 +/- 0.61, 2.18 +/- 1.19 and 3.28 +/- 1.09) were also higher than those of control group (0.62 +/- 0.37, 1.40 +/- 0.99 and 2.20 +/- 0.74; all P < 0.05). On day 9 after irradiation, the bone marrow CFU-GM yield of control group (19 +/- 11/10(6) cells) was significantly lower than that of treatment group (78 +/- 30/10(6) cells, P < 0.05). CONCLUSION: rhM-CSF expressed in silkworm could accelerate hematopoietic recovery in irradiated mice.

Animals↗

Transformation of Neomycin Resistance Gene (neo(R)) into Silkworm (Bombyx mori.L.).

Neomycin resistance gene (neo(R)) flanked by 5' and 3' regions of fibroin H-chain gene of silkworm (Bombyx mori.L.) was transferred into eggs of silkworm by gene gun in the early period of fertilization. The larvae were fed with an artificial diet containing neomycin in early 24 hours post transfettion, and some of them survived. The neo(R) encoding sequence in G(2) generation derived from the survivals was detected by Southern blotting. The results indicated that neo(R) could be used as a selective marker for studies on transgenic silkworm.

Journal Article↗

Studies on the Expression of the 28 kD Glutathione S-transferase Gene from Schistosoma japonicum in the Silkworm (Bombyx mori) Larvae and Insect Cells.

The gene of the 28 kD glutathione S-transferase (GST) from the Chinese strain of Schistosoma japonicum had been expressed in the silkworm (Bombyx mori) cells and larvae by Bombyx mori nuclear polyhedrosis virus (BmNPV) vector which had been modified. The GST gene was inserted into the right position of the BmNPV genome as identified by Southern hybridization. The product was a 28 kD protein, had GST activity and antigenicity. The yield in BmN cells (1x10(6) cells/ml) was 0.77 mg and more than 5 mg in a silkworm larvae. All the results showed that it is possible to develop the GST expressed in the silkworm larvae to a new kind of genetic engineered schistosomiasis vaccine.

Journal Article↗

PTTH--a potential growth activator in silkworm, Bombyx mori L. for enhancing silk production.

In silkworm, prothoracicotropic hormone (PTTH), directly or indirectly controls silk production and spinning activity along with juvenile hormone (JH). An effort was made to exploit the potential of PTTH by indirectly activating silk gland for increasing silk productivity using short chain synthetic analogues of PTTH. The analogy in action was also established using PTTH extracted from the silkmoth. Different doses of 42 synthetic PTTH analogues, viz., 2.5, 5, 10 and 20ppm and 3.3 mg/ml of PTTH extracted from silkmoth heads were administered orally to V instar silkworm larvae (Race:KAxNB4D2 and PMxNB4D2) at 0-144 hr at an interval of 24 hr. The analysed data showed an improvement of about 14 - 23% in KA x NB4D2 and about 10-14% in PMxNB4D2 in respect of cocoon shell weight on administration of some of the synthetic PTTH analogues. The PTTH extracted from the adult brain also showed similar effect. The structural analogy of synthetic PTTHs (which improved the shell weight) with original PTTH and its probable mode of action in silkworm are discussed.

Amino Acid Sequence↗

Molecular markers (RAPD) associated with growth, yield and origin of the silkworm, Bombyx mori L. in India.

To identify the molecular markers associated with growth and yield parameters in silkworm, Bombyx mori, RAPD profiles generated with seven UBC primers for fourteen silkworm stocks, originated from China, Japan, India and Russia, were statistically analyzed. Stepwise Multiple Regression Analysis establishes significant association of 45 markers with larval span, growth indices and four cocoon yield parameters relevant for silk production and t-test attest significance of the association of 89.5(1500 bp) and 54.13(300 bp), respectively with longer larval duration and high cocoon weight. The validity of this selection of markers was further supported with Discriminant Function Analysis (DFA) done on the basis of Mahalanobis D2 statistics. The two indices of yield/growth were also tested with DFA, which helped in identifying a few markers and thereby opened scope of using such marker (e.g. 91.11(900 bp)) for incorporating molecular markers in the breeding program for crop improvement in silkworm.

Animals↗

Molecular genetic approach for identifying markers associated with yield traits in the silkworm, Bombyx mori using RFLP-STS primers.

Bombyx mori, the mulberry silkworm, exhibits wide variability in yield and developmental attributes. The genetics of yield expression, shown to be of polygenic nature, is poorly studied in silkworm. To identify markers associated with 10 selected yield traits, multiple regression analysis (MRA) and discriminant function analysis (DFA) were applied on 64 markers generated with eight RFLP-derived sequence-tagged-site (STS) primers on the genomic DNA of 20 silkworm stocks of different origin and diverse yield potential. The analyses led to the identification of ten markers showing significant association with the different yield traits. The markers could classify the stocks according to yield potential, irrespective of their origin and status of diapause. Trait means were significantly different for stocks with and with out the associated marker. The inheritance of a marker G2(1300bp), selected at the first step of MRA for five yield traits was shown to segregate in 1:1 ratio in the F2 progeny from a cross between two divergent stocks. The relevance of the STS primers is discussed in the context of applying multiple regression model for identifying markers associated with yield expression and suitability for molecular breeding work in B. mori for yield improvement.

Animals↗