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.     

家蚕滞育卵与非滞育卵中几种关键酶活性的比较

家蚕Bombyx mori是卵滞育的昆虫, 在滞育期间无形态变化, 也不存在器官发育和组织分化, 然而其生理代谢过程仍在进行。为进一步研究家蚕滞育的机制, 本研究测定了家蚕滞育卵、 即时浸酸处理的滞育卵及非滞育卵在胚胎发育过程中的超氧化物歧化酶(superoxide dismutase, SOD, EC 1.15.1.1)、 过氧化氢酶(catalase, CAT, EC 1.11.1.6)、 丙酮酸激酶(pyruvate kinase, PK, EC 2.7.1.40)、 乙酰胆碱酯酶(acetylcholine esterase, AchE, EC 3.1.1.7)和乳酸脱氢酶(lactate dehydrogenase, LDH, EC 1.1.1.28) 的活性变化。结果表明： 处理后1-7 d, 即时浸酸处理的滞育卵, SOD活性由56 517.00 U/g提高到81 986.94 U/g, CAT活性由14.98 U/g提高到106.90 U/g, PK活性由25.19 U/g提高到181.70 U/g, AChE活性由17.88 U/g提高到287.86 U/g, 而LDH活性由169.96 U/g下降到122.82 U/g。 而在非滞育卵中, SOD活性由86 417.99 U/g下降到66 024.19 U/g, LDH活性由169.07 U/g下降到135.02 U/g; CAT活性由1.47 U/g提高到44.37 U/g, PK活性由20.56 U/g提高到92.09 U/g, AChE活性由21.40 U/g提高到99.17 U/g。在滞育卵中, SOD和AChE活性较稳定; CAT活性随发育上升, 而LDH活性随发育而下降; PK活性在胚胎发育的前 4 d呈上升趋势, 随后基本保持稳定。通过了解家蚕滞育卵、 非滞育卵与即时浸酸卵的相关酶活性在胚胎发育过程中存在的变化, 有助于进一步揭示家蚕滞育的机理。     

Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori.     

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm,Bombyx moil

To explore whether glutathione regulates diapause determination and termina tion in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapanse and nondiapauseegg producers, as well as those in dia pause eggs incubated at different temperatures. The activity ofthioredoxin reductase （TrxR） was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione （GSH/GSSG） ratio was observed in the ovary of diapauseegg producers, due to weaker reduction of oxidized glutathione （GSSG） to the reduced glutathione （GSH） catalyzed by glutathione reductase （GR） and TrxR. This indicates an oxidative shift in the glutathione redox cy cle during diapause determination. Compared with the 25℃treated diapause eggs, the 5℃treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.     

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。     

Injury‐induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori

Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho‐ERK levels and that injury increased the phospho‐ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation‐stimulated phospho‐ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation‐stimulated phospho‐ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c‐jun N‐terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho‐ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury‐induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs.     

Variation in glutathione status associated with induction and initiation of diapause in eggs of the bivoltine strain of the silkworm Bombyx mori

For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause.     

家蚕卵显微注射操作对蚕卵孵化及畸形蚕发生的影响

早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。     

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

N6‐methyladenosine (m⁶A) 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 m⁶A 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 m⁶A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m⁶A‐modification‐related gene expression and m⁶A content were greater in diapause‐destinated compared to nondiapause‐destined strains. Our findings suggest that m⁶A modification may provide significant epigenetic regulation of diapause‐related genes in the silkworm.     

家蚕细胞周期蛋白基因BmCcnl1的克隆及表达分析

家蚕Bombyx mori由受精卵到完成胚胎发育孵化的过程中, 细胞进行大量的分裂和分化, 然而滞育性卵的胚胎细胞分化至G2期便停滞在此阶段。为了探索这一发育阶段细胞内的分子调控, 本研究以人Homo sapiens的细胞周期蛋白基因cyclin L1为模板, 成功克隆了家蚕同源基因BmCcnl1（GenBank登录号: FJ889988）。BmCcnl1基因开放阅读框（open reading frame, ORF）全长1 254 bp, 编码417个氨基酸。利用Protean软件分析得出BmCcnl1蛋白预测分子量为49 kDa, 等电点为9.84。利用DNA重组技术构建了BmCcnl1基因的重组表达载体pET-21d-BmCcnl1, 对其进行原核表达, 其表达的蛋白以包涵体形式存在。利用RT-PCR技术分析了BmCcnl1基因在胚胎发育过程中的转录水平, BmCcnl1基因在非滞育性卵的胚胎发育阶段基本保持相对稳定的转录表达, 而滞育性卵从蛾体产下经过72 h后已经检测不到BmCcnl1基因的转录。结果提示, BmCcnl1基因与胚胎期滞育及非滞育性卵的发育调控相关。对该基因的克隆和表达分析为今后研究家蚕胚胎发育及细胞周期调控奠定了基础。     

即时浸酸显著提高滞育性家蚕卵辅酶Ⅰ和Ⅱ含量

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 提高了其呼吸耗氧量, 抑制了山梨醇积累。本研究利用HPLC法测定了家蚕滞育卵和5 min即时浸酸滞育性卵中辅酶Ⅰ和Ⅱ含量。结果表明： 产下后24-72 h, 家蚕滞育卵中NAD, NADH, NADP和NADPH含量分别下降了30%, 37%, 50%和4%; 而即时浸酸滞育性卵中分别增加了77%, 46%, 142%和241%。不过, 即时浸酸并未显著改变滞育性家蚕卵中NADH/NAD和NADPH/NADP比值。据此推测, 即时浸酸提高滞育性家蚕卵辅酶Ⅰ含量与其呼吸耗氧量增加有关; 即时浸酸显著提高辅酶Ⅱ含量与山梨醇积累抑制无关, 而主要与生物合成加强有关。     

Glutamine synthetase activity during embryonic and larval development of the silkworm Bombyx mori L. and role of a JH analogue

Abstract. Silkworm eggs of diapause nature were chilled or treated with hydrochloric acid. Glutamine synthetase activity in such treated eggs was present soon after the treatment, whereas in non-diapause eggs it was not detectable until 24 h after the start of development. During larval life, the glutamine synthetase was found to be absent in midgut tissue. Topical application of a JH analogue resulted in extened larval duration and it reduced glutamine synthetase activity initially, but in the latter part of development the activity was higher.     

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase     

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses

Abstract.  An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

家蚕体内维生素B6的存在形态和转换代谢

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori幼虫,探讨了家蚕体内维生素B（VB₆）化合物的存在形态和转换代谢途经。随饲料中盐酸吡哆醇（PN-HCl）添加量的增加,幼虫体内吡哆醇(PN)含量相应变化,其次是吡哆醛(PL);而辅酶型磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)含量存在稳定性。饲料中的吡哆醇以单纯扩散的形式进入体液;体液中的吡哆醇被各种组织吸收后,在各自的吡哆醛激酶和PNP/磷酸吡哆胺氧化酶的作用下,转变成辅酶型磷酸吡哆醛。家蚕不同于哺乳动物,没有特定的辅酶型磷酸吡哆醛形成组织和辅酶型磷酸吡哆醛的转送机制。同时家蚕体内缺乏具储存VB₆功能的辅酶型磷酸吡哆醛结合蛋白,推测这是用缺乏VB₆的饲料饲育各龄起蚕,幼虫当龄死亡的主要原因。     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.