Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Immune enhancement activities of silk lutein extract from Bombyx mori cocoons

Background

Declining immune function poses an important clinical challenge worldwide and supplementation with natural products that possessing immune enhancing properties is a promising approach for preventing or delaying immune function decline. Cocoons from yellow silkworms are a significant source of lutein, and this unexplored silk extract could be a viable alternative source for dietary lutein. This study assessed immunomodulatory activities of the silk lutein extract. Female BALB/c mice orally received lutein, either as silk or marigold extracts (10 or 20 mg/kg daily), or vehicle only (1% tween 80 in PBS pH 7.4) for 4 weeks. Natural killer (NK) cell activity, specific antibody production, lymphocyte subpopulations, mitogen-induced lymphocyte proliferation, and cytokine production were examined.

Results

Silk lutein extract increased NK cell activity, and the effect was dose-related whereas marigold lutein extract was ineffective. Silk lutein extract dose-dependently enhanced antibody production in pre-immunized mice but marigold lutein extract had no effect. Feeding with silk lutein extract increased the populations of CD3+ and CD4 + CD3 + cells. Silk lutein extract also stimulated concanavalin A- and lipopolysaccharide-induced proliferations of T and B lymphocytes, respectively. Moreover, silk lutein extract increased IL-2 and IFN-γ production while the effect of marigold lutein extract was undetectable.

Conclusions

Together, silk lutein extract enhanced both innate and adaptive immune functions. This preparation may prove to be an effective supplement for strengthened immunity.     

Biological relevance of host plant-derived terpenoid in the cocoons of the tropical tasar silkworm Antheraea mylitta

We have characterized and studied the biological functions of a terpenoid derivative in the Indian tropical tasar silkworm, Antheraea mylitta reared on the primary host plant Arjun, Terminalia arjuna. The compound from insect cocoon turned out to be a terpenoid derivative which resembled oleanane type triterpene (Arjunolic acid) present in the host plant. The plant and cocoon compounds were anti-oxidative as determined by bleaching of beta carotene in vitro. UV-exposure is the major form of peroxidative insult encountered by this wild tropical silkworm. The life cycle comprising five larval stages and the cocoon stage lasts for about 30–45 days. Hence the sequestration of antioxidant and UV-protectant molecule from the host plant commands great biological significance.     

珍贵绢丝昆虫——天蚕蚕蛾翅振行为观察

本文较系统地观察和测定了天蚕蚕蛾的翅振时间节律和翅振频率。结果表明,雄蚕蛾不论独处还是与雌蚕蛾共存,其翅振行为均出现于18：00至次日6：00之间,其中以22：00至次日4：00为主;雌蚕蛾独处时未见翅振行为,只有与雄蛾共存时方有此行为,出现于22：00至次日6：00。雌雄蚕蛾翅振行为参数,包括日翅振次数、翅振总时间、一次翅振持续时间、两次翅振间间隔时间等,因蚕蛾日龄和存在方式(独处或与异性共存)而异;雌雄蚕蛾相比,除两次翅振间隔时间外,雄蛾均明显高些。雌雄蚕蛾翅振频率一般为7-12Hz,不同日龄闯,雄蛾以羽化第2天为高,平均10.9741Hz;雌蛾以羽化第1天为高,平均10．7071Hz。同一日龄不同时刻间翅振频率有一定差异,但未达显著水平。此外,尚观察了雄蛾触角对其翅振行为表现的作用,最后讨论了制种中拟注意的有关问题。     

Green cocoons in silkworm Bombyx mori resulting from the quercetin 5-O-glucosyltransferase of UGT86, is an evolved response to dietary toxins

The glycosylation of UDP-glucosyltransferases (UGTs) is of great importance in the control and elimination of both endogenous and exogenous toxins. Bm-UGT10286 (UGT86) is the sole provider of UGT activity against the 5-O position of quercetin and directly influences the formation of green pigment in the Bombyx cocoon. To evaluate whether cocoon coloration evolved for mimetic purposes, we concentrated on the expression pattern of Ugt86 and the activities of the enzyme substrates. The expression of Ugt86 was not only detected in the cocoon absorbing and accumulating tissues such as the digestive tube and silk glands, but also in quantity in the detoxification tissues of the malpighian tubes and fat body, as well as in the gonads. As in the green cocoon strains, Ugt86 was clearly expressed in the yellow and white cocoon strains. In vitro, the fusion protein of UGT86 showed quercetin metabolic activity. Nevertheless, Ugt86 expression of 5th instar larvae was not up-regulated in the silk gland by exogenous quercetin. However, it was significantly up-regulated in the digestive tube and gonads (P < 0.05). A similar result was observed in experiments where larvae were exposed to rutin, an insect resistance inducer and growth inhibitor typically found in plants, and to 20-hydroxylecdysone (20E), an insect endocrine and plant source hormone. On the contrary, up-regulated Ugt86 expression was almost nil in larvae exposed to juvenile hormone III (P > 0.05). The results of HPLC revealed that a new substance was formed by mixing 20E with the recombinant UGT86 protein in vitro, indicating that the effect of Ugt86 on 20E was similar to that on exogenous quercetin derived from plant food, and that the effect probably initiated the detoxification reaction against rutin. The conclusion is that the reaction of Ugt86 on the silkworm cocoon pigment quercetin is not the result of active mimetic ecogenesis, but derives from the detoxification of UGTs.     

Detection and characterization of Wolbachia infection in silkworm

Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects.     

A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm,Antheraea pernyi

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.     

The cocoon spinning behaviour of the chinese oak silkworm, Antheraea pernyi

The cocoon of Antheraea pernyi is constructed in four successive phases, as resolved through movement recordings and time-lapse cinematography and cinefluorography: (1) scaffolding and peduncle (9·2 hr), (2) outer cocoon (13·9 hr), (3) cocoon impregnation (0·7 hr), and (4) inner cocoon (26·9 hr). The caterpillar reverses spinning direction at frequencies characteristic for each phase. The number of cycles (360-degree turns) within a phase is relatively constant from individual to individual, although the length of phase two varies seasonally. During cocoon impregnation the larva executes turns in rapid succession, ensuring the even distribution of a hindgut exudate which coats the cocoon with crystals and speeds tanning of the silk. In the second and fourth phases intracycle behaviour consists of extension-recovery loops of the anterior segments, each followed by a repositioning of the abdomen.     

高温对珍贵绢丝昆虫——天蚕卵巢生长发育的影响

结果表明,高温对天蚕（Ａｎｔｈｅｒａｅａ ｙａｍａｍａｉ）卵巢生长发育有明显的影响。３、４龄幼虫卵巢生长发育在２０￣２９℃范围内随温度提高而加快,３２℃下则略有下降;５龄幼虫卵巢生长发育在２９℃和３２℃下明显受阻。在茧蛹期,若在刚结茧或化蛹第１天即经受３２℃高温处理,卵巢大小及其可溶性蛋白含量多明显小于或低于２６℃,即其卵巢生长发育明显受阻;而在化蛹第６天开始受３２℃高温处理,结果则似反之。正因为     

Experimental sensitization with an extract of Dermatophagoides pteronyssinus ticks]

Immunogenic properties of Dermatophagoides pteronyssinus ticks were studied in subcutaneous injection of the antigen to guinea pigs and rabbits. Marked changes in the immunological reactivity of the animal organism were revealed; these changes were accompanied by production of agglutinating, sensitizing the mast cells and precipitating (in rabbits) antibodies, and by the development of hypersensitivity of the immediate and delayed type.     

Soil water dynamics in an oak stand

Our ability to predict toxic quantities of aluminium (Al) in acidic soils is limited by our understanding of the interactions between different solid forms of Al in solution and our lack of knowledge of which form control soluble Al. This review briefly considers each type of solid form of Al, particularly from a kinetic point of view and discusses models that have been developed to predict release of Al from individual forms. More comprehensive models (i.e. more than one source or sink of Al) are then discussed as well as the interactions between different solid sources of Al.     

Soil water dynamics in an oak stand

A model describing water uptake by plants with particular attention to the soil-root interface under transient conditions is derived and discussed. Field data on a daily scale enable the unknown parameters of the model to be determined with the help of an identification technique. The model is then used to analyse the experimental results presented in part I of this paper. The loss of total conductivity of the soil-tree system under drought conditions whereas the metabolism of the trees seems to remain unaffected can be explained by the increase of the soil-root resistance. In fact this resistance becomes the limiting factor when the volumetric soil water content decreases (below =0.33 for the superficial layer and 0.36 for the deeper one in the studied case). Such values can be frequently encountered at the end of summer.     

Cloning of the fibroin gene from the oak silkworm, Antheraea yamamai and its complete sequence

The nucleotide sequences containing an entire genomic region and 5 upstream region of Antheraea yamamai fibroin gene have been determined. The gene consists of an initial exon encoding 14 amino acids, an intron (150 bp), and a long second exon coding for 2641 amino acids. The fibroin coding sequence shows a specialized organization with a highly repetitive region flanked by non repetitive 5 and 3 ends. Northern blot analyses confirmed that fibroin gene is actively expressed in the posterior silk gland of the final instar larvae of Antheraea yamamai.     

Cytochemical study of the elimination chromatin in silkworm meiosis]

The elimination chromatin separating from chromosomes during the first maturation division of female sex cells of lepidoptera insects was studied cytochemically on paraffin sections of eggs of Bombyx mori L. Ocytes at the metaphase-telophase stage of the first meiotic division were stained for RNA by metyl green-pyronin and gallocyanin with negative results. These data differ from earlier positive results for Solenobia triquetrella reported by Ris and Kleinfeld, 1952.     

Phylogenetic analysis of Nosema antheraeae (Microsporidia) isolated from Chinese oak silkworm, Antheraea pernyi

The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU-ITS1-SSU-ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis.     

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.     

Detection of a cell-cycle inhibitor of Petunia hybrida protoplasts in an Arthrospira platensis extract

A chloroform crude extract of whole Arthrospira platensis cells, provisionally named A. platensis auto-inhibitor (APAI), was inhibitory to growth of human prostatic cells and was toxic to them at less than 25 g/ml. With cultured protoplasts of Petunia hybrida, APAI was growth inhibitory, without cytotoxicity, at 500 g/ml. Using flow cytometry, it was seen that APAI induced arrest of the plant cell division cycle at the G1 phase. © Rapid Science Ltd. 1998