Bombyx Eggs Research Articles

Expressions of the Protein Phosphatases PP1 and PP4 during the Embryonic Diapause Process of the Silkworm Bombyx mori.

Reversible protein phosphorylation is accomplished by the opposing activities of kinases and phosphatases. We previously demonstrated the regulation of serine/threonine protein phosphatase (PP) type 2A (PP2A) and 2B (PP2B or calcineurin) during the embryonic diapause process of Bombyx mori. In the present study, we further examine the expressions of other PPs (PP1 and PP4) during embryonic stages. An immunoblot analysis showed that Bombyx eggs contained a 38-kDa PP1 catalytic subunit (PP1-C), a 38-kDa PP4 catalytic subunit (PP4-C), and a 120-kDa PP1 nuclear targeting subunit (PNUTS), each of which underwent differential changes between diapause and developing eggs during the embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling diapausing eggs at 5°C for 70 days and then were transferred to 25°C, protein levels of PP1-C and PP4-C remained relatively high during the early embryonic stages and then decreased during middle (for PP1-C) or later (for PP4-C) embryonic stages. However, protein levels of PP1-C and PP4-C in diapause eggs remained at high levels during the first 8 days after oviposition. PNUTS protein levels showed inverse temporal changes, with increased levels being detected during the later embryonic stages of developing eggs. The direct determination of PP1 enzymatic activity showed higher activity in developing eggs than in diapause eggs. Examination of temporal changes in mRNA expression levels of PP1-C and PP4-C showed no difference between HCl-treated and diapause eggs. These results indicated that differential protein levels of PP1-C/PNUTS and PP4-C, and increased enzymatic activity of PP1 were likely related to the embryonic development of B. mori.

Expression of calcineurin in relation to the embryonic diapause process in the silkworm, Bombyx mori

Calcineurin (CN) is a Ca2+/calmodulin-activated serine/threonine protein phosphatase that is essential for translating Ca2+ signals into changes in cell function and development. In the present study, we investigated changes in CN expression during the process of embryonic diapause in the silkworm, Bombyx mori. An immunoblot analysis showed that Bombyx eggs contained a 59-kDa catalytic A subunit (CNA), a 19-kDa regulatory B subunit (CNB), and a 27-kDa calcipressin; the CNA, CNB, and calcipressin were found to undergo differential changes between diapause and developing eggs during the diapause process. In developing eggs, protein levels of CNA and calcipressin were high during the first stages and then gradually decreased with embryonic development. However, CNB protein levels showed inverse temporal changes, with increased levels being detected during later embryonic stages of developing eggs. In diapause eggs, protein levels of CNA and calcipressin remained at relatively high levels during the first 8 days after oviposition, but CNB levels remained at low levels. CN enzymatic activity was directly determined and results showed that it remained at low levels in diapause eggs during the first 8 days after oviposition. However, in developing eggs, CN enzymatic activity sharply increased during the first several days, reached a peak during middle embryonic development, and then greatly decreased 5 or 6 days before hatching. Examination of temporal changes in mRNA expression levels of CNB also showed differences between diapause and HCl-treated eggs. These results demonstrated that protein levels of CNA, CNB, and calcipressin, transcriptional levels of CNB, and CN enzymatic activity between diapause and developing eggs are differentially regulated, and these regulated changes are likely related to the embryonic diapause process of B. mori.

Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori

Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori.

Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination

For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 µmol/g fresh mass to 5-8 µmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 µmol/g fresh mass to 5.57 µmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination.

Chlorine distribution in the shells, and pH value of the contents from Bombyx eggs treated with HCl

Chlorine distribution in the shells, and pH value of the contents from Bombyx eggs treated with HCl

Dissociability of ribosomes in high salt buffer in relation to diapause of Bombyx eggs

Dissociability of ribosomes in high salt buffer in relation to diapause of Bombyx eggs

Esterase A4 elevation in cholesterol ester hydrolysis with respect to Bombyx egg diapause development

Esterase A4 elevation in cholesterol ester hydrolysis with respect to Bombyx egg diapause development

Egg extract promotes cell migration and growth in primary culture of early embryos in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Fetal bovine serum (FBS), commonly used in the culture of many insect cell lines, is a source of both nutrients and bioactive compounds that promote cell growth; however, because the growth-promoting compounds contained in FBS are not considered to be identical to those produced by insects, the response of insect cells to FBS is likely to differ from responses to endogenous growth-promoting compounds. The present study examined whether primary cultures of Bombyx embryonic tissue fragments respond differently to silkworm egg extract and FBS. It was observed that, in primary cultures, Bombyx egg extract promoted the migration and growth of cells from embryonic tissue fragments more potently than FBS. These results indicate that the extract of silkworm eggs in an early developmental stage contains molecules that strongly promote cell migration and growth. The use of egg extracts could also shorten the time required to establish a novel cell line.

Distinct effects of different temperatures on diapause termination, yolk morphology and MAPK phosphorylation in the silkworm, Bombyx mori

Previously, we showed that chilling of diapausing Bombyx eggs activated ERK/MAPK in yolk cells coincidentally with acquisition of developmental competence, and that ERK regulates diapause termination via activating transcription of key enzyme genes for ecdysteroid and sorbitol metabolism. To further elucidate the molecular mechanisms of temperature signaling and diapause termination, effects of different temperatures on diapause termination, yolk morphology, and ERK and p38 MAPK phosphorylation were analyzed. We confirmed the coincidence of diapause termination and changes of phosphorylated levels of ERK and p38 MAPK at various temperatures. Our study also shows that different temperatures have distinct effects on diapause termination and yolk morphology, however, these two effects did not always coincide. Our analyses suggest the possibility that the effects on diapause termination and yolk morphology are independently regulated by ERK. We observed that a temperature shift alters diapause intensity reversibly, and coincidental changes of phosphorylated ERK levels suggest that ERK may have a role in regulating the diapause intensity. The present study suggests that ERK and p38 MAPK have roles for transmitting the temperature signals that are important for diapause termination.

Fate mapping of the larval silk glands of Bombyx mori by UV laser irradiation of the egg at fertilisation.

Localised UV laser irradiation of Bombyx eggs at the fertilisation stage causes localised cuticle defects in the integument of the resultant larvae. Based on the correlation between the site of irradiation and the site of defects, I previously established a fate map of the larval integument of Bombyx mori. Although most of the internal organs of the irradiated specimens were eliminated in the acidic fixative used for cuticle preparation, silk proteins which had accumulated in the gland lumen were resistant to the acid treatment and silk gland morphology was thus preserved. Taking advantage of this phenomenon, I continued fate mapping, now of the larval silk glands of Bombyx mori, utilising the same specimens that were prepared and analysed in the previous study. Of 710 irradiated specimens analysed, 452 showed two normal glands, 27 showed one normal and one deformed gland, 21 showed one normal gland only, and 210 showed no silk gland. The sites of irradiation that caused the deletion of a silk gland were located within a limited area that entirely overlapped the ventral half of the area where irradiation caused defects in the labium. These results are consistent with previous histological descriptions stating that the silk glands develop from the paired invaginations in the labial segment. Based on the data, the presumptive region for the larval silk glands was located on the previously established Bombyx fate map.

Radiosurgery using heavy ion microbeams for biological study: Fate mapping of the cellular blastoderm-stage egg of the silkworm, Bombyx mori

We investigated the effects of heavy ions on embryogenesis of the silkworm, Bombyx mori using a collimated heavy ion microbeam from the vertical beam line of an AVF-cyclotron. Eggs were exposed to carbon ions at the cellular blastoderm stage. Microbeams were found to be extremely useful for radio-microsurgical inactivation of nuclei or cells in the target site. Spot irradiation caused abnormal embryos, which showed localized defects such as deletion, duplication and fusion, depending on dose, beam size and site of irradiation. The location and frequency of defects on the resultant embryos were closely correlated to the irradiation site. Based on this correlation, a fate map was established for the Bombyx egg at the cellular blastoderm stage.

Isolation, purification, and properties of cysteine proteinase from Bombyx mori L. eggs.

Silkworm moth (bombyx) egg cysteine proteinase with maximal activity at pH 3.0 was purified by chromatography and isoelectrofocusing. On SDS-electrophoresis in polyacrylamide gel the purified enzyme showed a single band of molecular mass 50 kD. Isoelectrofocusing revealed that the bombyx egg cysteine proteinase exists in two forms with pI values of 5.95 and 6.43, respectively. The enzyme activity was sensitive to inhibition by iodoacetamide and p-chloromercuribenzoate but resistant to EDTA, pepstatin, and phenylmethylsulfonyl fluoride. The cysteine proteinase hydrolyzes storage proteins of bombyx eggs but it was inactive with respect to N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA). It is a cathepsin L-like enzyme.

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-l-Phenylalanyl-l-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.

The profiles of mRNA levels for BHR39, a Bombyx homolog of Drosophila hormone receptor 39, and Bombyx FTZ-F1 in the course of embryonic development and diapause.

In the silkworm, Bombyx mori, the 5'-upstream region of the sorbitol dehydrogenase (SDH) gene contains nucleotide sequences that might be recognized and bound by steroid hormone receptors such as Drosophila hormone receptor 39 (DHR39) and Fushi tarazu transcription factor 1 (FTZ-F1). As a first trial to find out regulator(s) involved in gene expression for SDH in Bombyx eggs, we have cloned a cDNA encoding a Bombyx homolog (BHR39) of DHR39, and examined levels of mRNA expressions for BHR39 and Bombyx FTZ-F1 (BmFTZ-F1) during the entire embryonic development and diapause. During embryonic development of non-diapause eggs, mRNA expression for BmFTZ-F1, but not for BHR39, was shown to occur in parallel with that for SDH, although such a parallelism was not found at the termination of diapause, when SDH mRNA was expressed.

Molecular Cloning and Sequencing of cDNA That Encodes Cysteine Proteinase in the Eggs of the Silkmoth, Bombyx mori

We have isolated and sequenced a 1,486-base-pair near full-length cDNA coding for Bombyx egg cysteine proteinase. The cDNA encodes 344 amino acid residues containing a typical signal peptide sequence (16 residues), pro-peptide (104 residues), and the sequence for mature enzyme (224 residues). Sequence alignments show that the egg cysteine proteinase is similar to lobster cysteine proteinase (61% identity), barley cysteine proteinase, Aleurain (52%), rice cysteine proteinase, Oryzain (54%), and rat cathepsin L (59%). The amino-terminal sequencing of the egg cysteine proteinase indicates that the enzyme purified as an inactive form from eggs is a pro-enzyme. Pro-egg cysteine proteinase was detected in other silkmoth tissues such as ovary, fat body, hemocyte, and hemolymph by immunoblotting.

Cysteine proteinase from Bombyx eggs: Role in programmed degradation of yolk proteins during embryogenesis

Cysteine peptidases are involved in physiological processes of insect development and have been considered as potential targets for the development of insect control strategies. In this study, we obtained a recombinant cysteine cathepsin L (AsCathL) from leaf-cutting ant (Atta sexdens), a species from the order Hymenoptera who causes enormous damage to crops, natural forests and reforested areas. RT-qPCR showed AsCathL expression throughout insect development and in all body parts of the adult insect analysed, suggesting its role as a lysosomal cathepsin. AsCathL encodes a protein of 320 amino acid residues consisting of a pro-peptide and the mature with amino acids sequence over 67% similarity with lysosomal cathepsin L of species from Lepidoptera and Diptera. Phylogenetic tree revealed that AsCathL is very similar to predicted cathepsins found in other ants. Recombinant AsCathL was expressed in insoluble form by Escherichia coli Arctic Express (DE3) RIL, purified under denaturing conditions and refolded. The enzyme showed hydrolytic activity in vitro towards synthetic substrate Z-Phe-Arg-AMC at acidic pH. Synthetic inhibitor E−64 acted against peptidase activity and a study regarding the interaction between E−64 and AsCathL using nuclear magnetic resonance (NMR) revealed that 83.18% from all E−64 molecules are irreversibly bound to AsCathL. In addition, the proteolytic activity of AsCathL was strongly inhibited by recombinant sugarcane cystatins with Ki ranging from 0.6 nM to 2.95 nM. To the best of our knowledge this is the first report characterizing a cysteine peptidase from leaf-cutting ants, which may contribute to future studies of ants’ cathepsins.

Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5°C in diapause eggs of the silkworm, Bombyx mori

1. 1. In diapausing eggs of the silkworm, Bombyx mori, activity of NAD-sorbitol dehydrogenase (EC 1.1.1.14, SDH) is almost negligible, but is increased by acclimation at 5°C (Yaginuma et al., 1990, J. comp. Physiol. B160, 277–285). To elucidate the mechanism regulating SDH activity, the following experiments were conducted. Anti-SDH serum was made in a mouse using purified sheep liver SDH. 2. 2. This antiserum reacted with Bombyx egg SDH purified partially by Blue Sepharose CL-6B and Sephacryl S-300 column chromatographies. 3. 3. SDS-PAGE and immunoblotting analyses using the antiserum showed that SDH activity was correlated with the amount of the enzyme protein. 4. 4. These results indicate that biosynthesis of SDH is induced by acclimation at 5°C in diapause eggs of B. mori.

Comparison of egg and embryo proteins and a trial to detect proteolytic activities in eggs of Bombyx mori

1. 1. One- or two-dimensional gel electrophoresis of proteins in the Bombyx eggs at various stages revealed spots corresponding to vitellin heavy chain (VH), vitellin light chain (VL), egg specific proteins (ESPs) and 30 kDa proteins. 2. 2. Isolated embryos showed many other minor components and abundant proteins at 30 kDa. 3. 3. Incubation of crude egg extracts at pH 3 and 4 at 37°C caused a rapid decrease of electrophoresis spots corresponding to VH, VL and ESP. 4. 4. At pH 5, a preferential decrease of ESP spots was seen. 5. 5. Under all acid, neutral and alkaline conditions tested proteins remained intact.

Quantitative and qualitative changes of polypeptides of egg-specific protein and vitellin depending on the diapause state of Bombyx eggs exposed to lower temperatures

Quantitative and qualitative changes of polypeptides of egg-specific protein and vitellin depending on the diapause state of Bombyx eggs exposed to lower temperatures

Control of polyol metabolism and diapause duration of Bombyx eggs by anaerobiosis

Control of polyol metabolism and diapause duration of Bombyx eggs by anaerobiosis