Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

Investigation of the molecular mechanism underlying the inhibitory activities of ethanol extract of Bombyx mori pupa-incubated Cordyceps militaris fruiting bodies toward allergic rhinitis.

Cordyceps militaris has been widely studied for its various pharmacological activities such as antitumor, anti-inflammation, and immune regulation. The binding of an allergen to IgE-sensitized mast cells in nasal mucosa triggers allergic rhinitis. We found that oral administration of 300 mg/kg of the ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruiting bodies significantly alleviated the symptoms of ovalbumin-induced allergic rhinitis in mice, including sneeze/scratch, mast cell activation, eosinophil infiltration, and Syk activation. The treatment of ethanol extract significantly suppressed the release of ß-hexosaminidase (a degranulation marker) and mRNA expression levels of various cytokines, including IL-3, IL-10, and IL-13 in activated RBL2H3 cells. The ethanol extract and ß-sitostenone, which was purified from the extract, could respectively reduce the Ca2+ ion mobilization in activated RBL-2H3 cells. Furthermore, results collected from western immunoblotting demonstrated that ethanol extract significantly retarded Ca2+ ion mobilization-initiated signaling cascade, which provoked the expression of various allergic cytokines. Also, the extract incubation interfered with P38 as well as NF-kB activation and Nrf-2 translocation. Our study suggested that ethanol extract possessed some natural constituents which could inhibit immediate degranulation and de novo synthesis of allergic cytokines via inhibition of Ca2+ ion mobilization in mast cells in the nasal mucosa of allergic rhinitis mice.

A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori.

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

Pupae protein extracts exert anticancer effects by downregulating the expression of IL-6, IL-1ß and TNF-α through biomolecular changes in human breast cancer cells.

The Pupae of Bombyx mori and Samia ricini are a source of high-quality proteins and essential nutrient elements for human. Recent studies revealed that protein extracted from pupae possessed therapeutic benefits for the treatment of many diseases. However, the anticancer activity of protein extracts from the pupae of B. mori and S. ricini has been rarely reported. Our objective was to study the effect of protein extracts from the pupae of B. mori and S. ricini on cytotoxicity and expression of pro-inflammatory cytokines; IL-6, IL-1ß and TNF-α, in breast cancer cells (MCF-7). Additionally, anticancer action of protein extracted from the pupae was further investigated through biomolecular changes in MCF-7 cells using Fourier transform infrared (FTIR) spectroscopy. Pupae protein extracts of B. mori exhibited cytotoxic effects with an IC50 value of 15.23 + 0.4 µg/mL with higher selectivity than doxorubicin on MCF-7 cells. Fourier transform infrared (FTIR) spectroscopy revealed that lipid contents in MCF-7 cells treated with pupae protein extracts of B. mori were higher than untreated cells. Treatment with protein extracts from pupae of B. mori or S. ricini caused significantly reduced protein and nucleic acid contents of MCF-7 cells. The expression of IL-6, IL-1ß and TNF-α in MCF-7 treated cells was investigated using RT-qPCR and ELISA. Our results revealed that protein extracts from the pupae of B. mori or S. ricini significantly decreased IL-6, IL-1ß and TNF-α in MCF-7 cells both at mRNA and protein levels. Expression of IL-6 and IL-1ß in MCF-7 treated cells, especially IL-6, was strongly reduced compared to untreated cells, while TNF-α expression was slightly decreased. These findings suggest that pupae protein extracted from B. mori or S. ricini may play a role in breast cancer through a down-regulatory action on the expression of IL-6, IL-1ß and TNF-α, and may also exert anticancer effects by causing biochemical changes of lipids, proteins and nucleic acids. These findings indicate that pupae protein extracted from B. mori or S. ricini may provide a potential novel therapeutic target for breast cancer.

Estrogen-related receptor participates in regulating glycolysis and influences embryonic development in silkworm Bombyx mori.

Estrogen-related receptors (ERRs) play indispensable roles in development, energy metabolism, and cancers and are metabolic switches in Drosophila. However, the mechanism underlying their metabolic role is unknown in insects. This study analysed the expression profiles of Bombyx mori ERR (BmERR), hexokinase (BmHK), pyruvate kinase (BmPK) and phosphofructokinase (BmPFK) during embryonic development. The expression of BmERR tended to be similar to that of the other genes. We observed a regulatory association between BmERR and glycolytic rate-limiting enzymes by BmERR overexpression, RNA interference (RNAi), and ERR inhibitors in B. mori embryo cells. Subsequently, ERR cis-regulation elements (ERREs) were predicted and identified in the BmPFK promoter. Transfection assays, electrophoretic mobility shift assays and chromatin immunoprecipitation showed that BmERR can bind to one of these elements to regulate the expression of BmPFK. ERREs were also predicted in the BmHK and BmPK promoters. In the eggs, the expression of glycolytic rate-limiting enzyme genes was suppressed when the expression of BmERR was interference by double-stranded BmERR, the glucose levels also was increased. Meanwhile, the development of silkworm embryos was delayed by about 1 day. These results indicate that BmERR can bind to the ERREs of glycolytic gene promoters and regulate the expression of glycolytic genes, ultimately affecting embryonic development in silkworms.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori.

Steroid hormones, represented by estrogen and testosterone, act as sex hormones that play an essential role in the sexual differentiation of vertebrates. However, it remains unclear whether ecdysteroids, typical steroid hormones in insects, function as sex hormones. In this study, we investigated whether ecdysteroids or ecdysone signals are involved in the sexual differentiation of the silkworm (Bombyx mori) embryo. Quantitative analysis using LC-MS/MS demonstrated that there was no significant difference in the 20-hydroxyecdysone (20E) titer between sexes during embryonic development. Consistent with this result, expression levels of 2 genes encoding ecdysteroid-phosphate phosphatase (EPPase) and ecdysone 20-hydroxylase (E20OHase), which are essential for the biosynthesis of ecdysone and 20E in eggs, did not show a significant difference between male and female embryos. Expression levels of ecdysone receptor (EcR) and E75, which is one of a small set of genes induced directly by 20E, were also similar between the 2 sexes. However, knockdown of EPPase and one isoform of EcR (EcR-A) resulted in decreased expression of Bombyx doublesex (Bmdsx), a master regulatory gene for sexual differentiation of the silkworm in both male and female embryos. In vitro analysis with cultured testes revealed that expression levels of Bmdsx were increased in a dose-dependent manner of the ecdysone analog, ponasterone A. These results suggest that ecdysone signaling may play a role in indirectly regulating the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx in the silkworm.

Bombyxin/Akt signaling in relation to the embryonic diapause process of the silkworm, Bombyx mori.

Our previous study showed that phosphorylation of glycogen synthase kinase (GSK)-3ß is related to the embryonic diapause process in Bombyx. However, the upstream signaling pathway was not clearly understood. In the present study, we examined bombyxin/Akt signaling in relation to the embryonic diapause process of B. mori. Results showed that GSK-3ß phosphorylation stimulated by dechorionation was blocked by LY294002, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating involvement of PI3K in GSK-3ß phosphorylation in dechorionated eggs. Direct determination of Akt phosphorylation showed that dechorionation stimulated Akt phosphorylation. The Akt phosphorylation was blocked by LY294002. Temporal changes in Akt phosphorylation showed that different changing patterns exist between diapause and developing eggs. Relatively higher phosphorylation levels of Akt were detected between days 3 and 5 after oviposition in non-diapause eggs compared to those at the same stages in diapause eggs. Upon treatment with HCl, which prevents diapause initiation, Akt phosphorylation levels exhibited a later and much broader peak compared to diapause eggs. Examination of expression levels of the bombyxin-Z1 gene showed that in diapause eggs, a major peak occurred 1 day after oviposition, and its level then sharply decreased on day 2. However, in both non-diapause and HCl-treated eggs, a major broad peak was detected between days 1 and 4 after oviposition. These temporal changes in bombyxin-Z1 gene expression levels during embryonic stages coincided with changes in Akt phosphorylation, indicating that bombyxin-Z1 is likely an upstream signaling component for Akt phosphorylation. Taken together, our results indicated that PI3K/Akt is an upstream signaling pathway for GSK-3ß phosphorylation and is associated with the diapause process of B. mori eggs. To our knowledge, this is the first study to demonstrate the potential correlation between bombyxin/Akt signaling and the embryonic diapause process.

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.

Vertebrate estrogen regulates the development of female characteristics in silkworm, Bombyx mori.

The vertebrate estrogens include 17-ß-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.

Female qualities in males: vitellogenin synthesis induced by ovary transplants into the male silkworm, Bombyx mori.

Female qualities in males are common in vertebrates but have not been extensively reported in insects. Vitellogenin (Vg) is highly expressed in the female fat body and is generally required for the formation of yolk proteins in the insect egg. Vg upregulation is generally regarded as a female quality in female oviparous animals. In this study, we found that Bombyx mori Vg (BmVg) is especially highly expressed in the female pupa. Downregulation of the BmVg gene in the female pupa by RNA interference (RNAi) interfered with egg formation and embryonic development, showing the importance of BmVg in these processes. So, we used BmVg as a biomarker for female qualities in the silkworm. Hematoxylin-eosin staining and immunofluorescence histochemistry showed that ovary transplants induced BmVg synthesis in the male pupa fat body. Ovaries transplanted into male silkworms produced only a few eggs with deformed yolk granules. These results suggested that the amount of BmVg in the male silkworm was insufficient for eggs to undergo complete embryonic development. After 17-beta-estradiol was used to treat male pupae and male pupal fat bodies, BmVg was upregulated in vivo and in vitro. These findings indicated that the male silkworm has innate female qualities that were induced by a transplanted ovary and 17ß-estradiol. However, in silkworms, female qualities in males are not as complete as in females.

Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori.

In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼ 21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

[Study of intracellular localization of the proteolytic enzyme complex and its protein inhibitors in bombyx grain].

Intracellular localization of serine, cysteine and aspartate proteases, as well as their protein inhibitors, in bombyx grain in the postdiapause period of embryogenesis has been studied. Proteolytic activity of aspartate and cysteine proteases was found in lysosomal, mitochondrial, and nuclear fractions of grains. Serine protease activity was not observed in subcellular fractions of grains of the fourth day of postdiapause development. It has been shown that activities of protein inhibitors and certain peptide hydrolases in subcellular fractions provide consistent functioning and fine regulation of the proteolytic enzyme complex.

[Chromatin in diapause of the silkworm Bombyx mori L.: thermal parthenogenesis and normal development].

Having used hematoxylin as a stain, some features of silkworm embryo chromatin in diapause have been studied in normal and parthenogenetic development. With found direct correlation between the number of interphase chromatin grains and the number of chromosomes in the nucleus, we examined cell polyploidization in the embryo at diapause stage. Polyploidization by parthenogenesis is not reducible to endomitotic doubling of the chromosome set because it comprises 6n-nuclei. Explanation of more diverse range of polyploid cells in parthenogenesis needs to consider the fusion of cleavage nuclei that is carried out by the cytoplasmic karyogamic mechanism in the absence of fertilization. For the first time on squash preparations, in diapausing embryo, we have identified primary germ cells (PGC) that are characterized by less compact chromatin, especially in the zygotic form of development, a larger size of the nucleus and cytoplasm, and irregular number and size of nucleoli. Evaluation of PGC ploidy in parthenogenesis by calculation of "loose" chromatin grains in diapause is possible and testifies polyploidization in embryo germ-line. This explains the inevitable admixture of tetraploid eggs in diploid parthenoclone grain and its absence in normal development. Cytological method used has revealed a spiral arrangement of chromatin grains on the inner surface of the nucleus at different levels of ploidy.

Differential expression of the Bombyx mori diapause-termination timer gene Ea4 in diapause-inducing temperature and photoperiod.

Diapause in Bombyx mori eggs is induced by temperature and photoperiod at the stage of embryonic development in the maternal generation. In those diapause eggs, Esterase-A4 is suggested to serve as a diapause-termination timer (TIME-EA4), because its ATPase activity shows an interval-timer elevation after acid treatment or chilling of eggs to break diapause. To clarify whether the timed ATPase activity of TIME-EA4 is related to its gene (Ea4) expression, we analyzed Ea4 mRNA of eggs in diapause-inducing environmental conditions. Reverse transcription PCR (RT-PCR) analysis showed that the level of Ea4 mRNA was lower in 15DD than in 25LL (P < 0.01) or in 20LD (P < 0.01) but did not oscillate when photoperiod and temperature periodically oscillated (P > 0.05). Furthermore, expressed sequence tag profile and gene microarray analysis demonstrated that Ea4 showed stage-specific and tissue-specific expression during postembryonic stages, high Ea4 mRNA in the spinning and eclosion stages, and in integument and head, but low in gonads of fifth-instar day-3 larvae. Then we analyzed the relationship between TIME-EA4 ATPase activities and Ea4 gene expression. The ATPase activities in diapause eggs laid by the resultant adults transiently elevated after treatments to break diapause, that is, at 1.5 h after common-acid treatment and day 13 after chilling at 5°C. However, these elevations of enzyme activities were not accompanied by any increases in Ea4 mRNA levels. In conclusion, the termination of the Bombyx embryonic diapause is related to TIME-EA4 but not to its gene expression.

Identification and characterization of Polycomb group genes in the silkworm, Bombyx mori.

Polycomb group (PcG) proteins are involved in chromatin modifications for maintaining gene repression that play important roles in the regulation of gene expression, tumorigenesis, chromosome X-inactivation, and genomic imprinting in Drosophila melanogaster, mammals, and even plants. To characterize the orthologs of PcG genes in the silkworm, Bombyx mori, 13 candidates were identified from the updated silkworm genome sequence by using the fruit fly PcG genes as queries. Comparison of the silkworm PcG proteins with those from other insect species revealed that the insect PcG proteins shared high sequence similarity. High-level expressions of all the silkworm PcG genes were maintained through day 2 to day 7 of embryogenesis, and tissue microarray data on day 3 of the fifth instar larvae showed that their expression levels were relatively low in somatic tissues, except for Enhancer of zeste (E(Z)). In addition, knockdown of each PRC2 component, such as E(Z), Extra sex combs (ESC), and Suppressor of zeste 12 (SU(Z)12), considerably decreased the global levels of H3K27me3 but not of H3K27me2. Taken together, these results suggest that insect PcG proteins are highly conserved during evolution and might play similar roles in embryogenesis.

Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori.

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH(2) (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, ß- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-ß-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.

Characterization of Bombyx embryo segmentation process: expression profiles of engrailed, even-skipped, caudal, and wnt1/wingless homologues.

To gain insight into segmentation processes, the expression at embryonic stages of the silkmoth Bombyx mori homologues of even-skipped (eve), engrailed (en), caudal (cad), and wnt1/wingless (wg) transcripts were examined by whole mount in situ hybridization. Pair-rule eve stripes and segmental en and wnt1/wg stripes were generated sequentially from anterior to posterior, confirming the previous results that showed that Bombyx belongs to short-germ insects. However, unlike in previously described short germ insects, the segmentation of Bombyx occurred without marked germ band elongation: the putative growth zone was expanded compared with previously described short germ insects. This may indicate that Bombyx represents an evolutionarily intermediate state in a transition from short to long germ type. The expressions of cad and wnt1/wg, which are known to be present in the growth zone in short germ insects, initially showed a large median expression domain that, as segmentation proceeded, later retracted to the posterior pole. This is also unique to this insect. Detailed analysis of their relative expressions indicated that wnt1/wg domain retracted faster than the cad domain, and double stain in situ hybridization suggested that the eve stripe appears from cells that have ceased to express wnt1/wg. Another unique aspect of Bombyx embryogenesis is that gastrulation began at later embryonic stage compared with other insects and proceeded slowly from anterior to posterior. On the basis of these results, conserved and divergent aspects of the evolution of insect segmentation mechanisms and germ cell formation are discussed.

Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae.

With manganese superoxide dismutase (SOD) expressed in silkworm larvae, Bomby mori L, we investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose-dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.

Biomolecular changes and somatic mutations induced by UV laser irradiation at embryonic stage of Bombyx mori.

PURPOSE: To determine the impact of ultraviolet (UV) laser radiation on Bombyx mori embryos in terms of its effect on embryonic and larval haemolymph proteins and morphological traits. MATERIALS AND METHODS: The eggs of silkworm strain NB4D2 were exposed to third harmonic laser pulses at 355 nm from a Nd:YAG laser for different durations of 30, 60, 90, 120, 150, or 180 sec. Morphological changes induced by the UV laser were analysed at larval, pupal and adult stages. The eggs exposed to UV laser irradiation at different developmental stages were subjected to protein analysis by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph derived from irradiated and control larva was also analysed by SDS-PAGE. RESULTS: UV laser irradiation resulted in various structural polymorphisms. Asymmetrical fusion of segments was not confined to larva but persisted throughout pupal and adult stages. Development of extra caudal horn, unequal size and lack of antenna, retarded thoracic legs and variation in larval markings were observed. Comparatively, the effect of the UV laser on 8- and 16-h old embryo was greater than on the other stages. The changes in protein pattern were not distinct until the 5th day of embryogenesis as revealed by SDS-PAGE. A 178 kiloDalton (kDa) protein resolved into 198, 184 & 169 kDa polypeptides and 154 kDa new protein band along with other proteins of 110, 45, 41 & 38 kDa were noticed in irradiated eggs at the 6th day. Further, 33, 32, and 6.2 kDa new protein bands were observed in the haemolymph of 5th instar silkworm larvae derived from UV laser irradiated embryos. CONCLUSIONS: A comparative analysis of the present study revealed that UV laser not only induced continuous structural polymorphisms (somatic mutations), but also induced protein changes with the appearance of new protein bands in embryonic and haemolymph protein. The UV laser could be a potential tool for biochemical genetics and genome analysis in B. mori.