The consumption and utilization of the spiral blue-green algae Spirulina platensis in artificial diets by the silkworm, Bombyx mori

The consumption and utilization of various concentrations of the spiral blue-green algae, Spirulina platensis (Nordst.) Geitl., by the fourth-instar larvae of the silkworm, Bombyx mori L., was studied by incorporation into an artificial diet. The silkworms ingested more food on the low algal-powder diets, whereas they digested more efficiently on the high algal levels. The larvae fed on diets containing 30–40% algae had the shortest larval duration compared to those on low algal-powder diets. The freeze-dried powder of S. platensis is well utilized by B. mori larvae when added to artificial diets at levels between 15% and 30%, although the high algal content at 40% was slightly better for the efficiency of conversion of ingested diet into body substance (E.C.I.) and the cocoon production. However, analysis of the protein content in feces indicated an excess of the algal powder at 40%. Measurements of data on the food consumption and utilization in this study proposed that this spiral alga is dietetically utilizable as a protein source for rearing the silkworm in smaller quantities than on a soybean meal.

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Resúmé La consommation et l'utilisation de différentes concentrations de Spirulina platensis par les chenilles de 4ème stade de Bombyx mori, ont été étudiées par incorporation dans un aliment artificiel. Les vers à soie ont ingéré plus d'aliment avec les régimes à faible concentration en algue, tandis qu'ils ont digéré plus efficacement les régimes à haute concentration d'algue. Les chenilles alimentées sur des régimes contenant 30 à 40% d'algue ont eu un développement larvaire plus bref que celles alimentées sur des régimes à faible concentration. La poudre congelée et séchée de S. platensis incorporée aux taux de 15 à 30% dans le régime artificiel est bien utilisée par les chenilles de B. mori, quoiqu'une teneur de 40% soit légèrement meilleure pour l'efficacité de conversion de l'aliment en matière vivante (E.C.I.) et la production de cocons. Cependant, l'analyse du contenu protéique dans les excréments révélait un excès de poudre d'algue à 40%. L'analyse des données au cours de cette étude, sur la consommation et l'utilisation des aliments, montre que S. platensis est utilisable diét étiquement en plus petite quantité que la farine de soja comme source de protéines pour l'élevage du ver à soie.

     

Hierarchical clustering of 54 races and strains of the mulberry silkworm,Bombyx mori L: Significance of biochemical parameters

Summary A detailed analysis was undertaken to test the efficacy of hierarchical agglomerative clustering (UPGMA method) in grouping the races and strains of the mulberry silkworm, Bombyx moti L., and to ascertain the importance of biochemical parameters in the clustering process. The analysis was based on data from two rearing seasons with 54 selected races/strains of different geographic origin and varying yield potentials. The results indicate that seven clusters can be realised with yield parameters alone, whereas the inclusion of biochemical parameters in clustering resulted into two broad groups: one having all the breeds with high cocoon weight and shell weight, the other having all the low-yielding silkworm strains both from India and from other countries. Further sub-grouping under these two groups highlights genetical differences associated with the differentiation of various groups of races in temperate and tropical areas as well as their significance for silkworm breeding. Estimates of all ten variables were further subjected to quick clustering and the results showed that cluster 5, constituted by 38 lowyielding strains of India, China and Europe, had the highest values of the final cluster centre for amylase and the effective rate of rearing (ERR), while clusters 1 and 4 had the highest values for invertase and alkaline phosphatase. The evolutionary aspect of the genetic channelisation of silkworm races from various countries is discussed against the background of differences in the biochemical parameters and yield variables.     

三眠蚕诱导剂咪唑类物质KK-42对桑蚕内分泌系统的作用

为了研究作为三眠现诱导剂的一种唑化合物RK-42的作用机制,本文应用侧休(CA)短期体化学测定Bombyx motx则的活性,通过记蟆皮(MH)的垫射免疫分析(RIA)法测定了血苏巴内根据的放射免疫法成功珍KK-42作用后的肢导泌侃前胸蚊腺面的试验,提出了KK-42作用的新解说,吧调体活性的湖汇KK-42与侧体外支明KK-42应用下桑蚕四龄前期时,把器官首先见侧休,咽侧体看成性反使用分泌PTTH产生相应变化,从而引起蜕皮激素高峰的推迟.推迟了的蜕皮素峰与扣对馈管的保幼激素JH共同作用的结果引起了化蛹蜕皮,产生了三眠蚕.     

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.     

The amino acid/K symporters for neutral amino acids along the midgut of lepidopteran larvae: Functional differentiations

The functional properties of the K⁺-dependent symporter for neutral amino acids have been investigated in brush border membrane vesicles prepared from the anterior, middle and posterior portions corresponding to the three morphologically distinguishable regions of the midgut of Bombyx mori larvae. An intravesicular accumulation of leucine was driven by a K⁺-gradient in the three preparations, but vesicles from the posterior tract displayed much higher uptake and accumulation values. Kinetic analysis of leucine uptake, performed in experimental conditions which mimic as closely as feasible experimentally those occurring in vivo (Δψ = −90 mV, pH_in7.2/pH_out8.7, [K⁺]_out100 mM), evidenced that the affinity for the amino acid was similar along the midgut (150 μM), but V_max in the posterior region was more than 11-fold higher than that of the anterior-middle tract (11.3 ± 0.7 and 0.98 ± 0.07 nmol/7s/mg protein, respectively). Leucine uptake was remarkably influenced by extravesicular pH and by Δψ only in vesicles from the posterior midgut: a lowering of pH to 7.2 caused a sevenfold increase of K_m, whereas in the absence of Δψ, V_max decreased threefold. The selectivity sequence for the alkali cations was somewhat different in the two midgut regions, but K⁺ remained the most effective. In the posterior midgut, the selectivity for K⁺ was greatly enhanced when a transmembrane electrical potential was present. Leucine kinetics as a function of external potassium concentration was hyperbolic in the posterior and sigmoidal in the anterior-middle part. Inhibition of leucine uptake induced by a 20-fold excess of different amino acids suggested the presence in both midgut tracts of a broad specificity system for neutral amino acids, with many-but not all-features in common with the B^o system of mammal intestinal and renal epithelial brush borders. However, there are differences between the two midgut regions as regard to the ability of the symporters to recognize the different amino acids, which concern the side chain and the presence of the aromatic ring. Altogether these data suggest that two kinds of symporters for neutral amino acids, with different functional properties, are expressed in the anterior-middle and posterior regions of the lepidopteran midgut.     

Genetically identical copies of the mulberry silkworm

Summary Genetically identical copies of the silkworm female obtained via ameiotic parthenogenesis are insignificantly depressed by artificial reproduction, and notwithstanding their isogenous pattern, they show the same variability in quantitative characters as in the heterogenous forms.By means of androgenesis, genetically identical copies of strictly homozygous males were obtained; the latter were produced via meiotic parthenogenesis. The androgenetic clones of these males were transformed by backcrosses into homozygous bisexual lines. Viability and cocoon weight in these lines were higher than those in androgenetic clones of similar genotype, but they were far from the norm due to the depressing effect of semilethals left in them in a homozygous state. An extremely low phenotypical variability of quantitative characters is observed in the isogenous hybrids F₁ obtained from crossing two genetically dissimilar strictly homozygous individuals. These hybrids make excellent material for phenogenetical studies. The female and male isogenous clones have an increased combining ability which is acquired in the course of their selection for high disposition towards complete parthenogenesis. For practical purposes it is proposed to take a female and a male which when mated produce a high-quality family and clone them separately, the first via parthenogenesis, the latter via androgenesis. After mating these reproduced bisexual clones, it will be possible to obtain in successive generations a vast number of families repeating the prominent productivity of the initial family.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase     

家蚕抗真菌因子BmSPI39对球孢白僵菌入侵的表达响应

【目的】制备家蚕Bombyx mori抗真菌因子BmSPI39的多克隆抗体,分析BmSPI39对球孢白僵菌Beauveria bassiana入侵的表达响应。【方法】利用原核表达和固定化镍离子亲和层析技术获得高纯度的BmSPI39重组蛋白,利用胶内活性染色检测重组蛋白BmSPI39对枯草杆菌Bacillus subtilis蛋白酶A的抑制活性;利用重组蛋白BmSPI39,通过免疫新西兰大白兔制备BmSPI39的多克隆抗体。基于家蚕开放性数据库SilkDB 3.0,分析BmSPI39在4龄第3天至化蛹后1 d家蚕免疫相关组织体壁、中肠和脂肪体中的时空表达特征。利用制备的BmSPI39抗体通过Western blot分析感染球孢白僵菌不同时间后家蚕幼虫和蛹体壁中BmSPI39应答球孢白僵菌入侵的表达响应。【结果】胶内活性染色结果显示,重组BmSPI39蛋白能够强烈抑制枯草杆菌蛋白酶A的活性。酶联免疫吸附结果显示,BmSPI39抗血清的抗体效价达1∶128 000。Western blot和胶内活性染色结果显示,纯化后的BmSPI39抗体能够有效结合不同多聚体化状态下的BmSPI39抗原蛋白。BmSPI39表达数据的热图分析显示,BmSPI39在游走期和预蛹期的家蚕体壁中有表达,在预蛹期的脂肪体中的表达量较高,但在各发育阶段的中肠中均不表达。Western blot分析结果表明,家蚕幼虫体壁中的BmSPI39的表达在感染球孢白僵菌后84 h上调表达,108和120 h下调表达。【结论】本研究成功制备了BmSPI39的多克隆抗体。BmSPI39蛋白的表达能够响应球孢白僵菌入侵,可能参与蛹期家蚕抵御真菌入侵的过程。     

家蚕5龄幼虫精巢和卵巢microRNA芯片及转录组比较分析

【目的】本研究旨在通过对家蚕Bombyx mori 5龄幼虫精巢和卵巢组织微小RNA (microRNA, miRNA)基因芯片及转录组进行分析,找到参与家蚕性腺发育相关的miRNA分子及可能的靶基因。【方法】采用新一代高通量测序平台对家蚕5龄幼虫精巢和卵巢(分别定义为Test和Control)进行miRNA基因芯片检测及转录组测序分析,根据P＜0.05且log₂(fold change, FC)≥2的标准,通过比较筛选出Test vs Control的差异表达miRNA;根据q≤0.05且|log₂(fold change)|≥1的标准,通过比较筛选出Test vs Control的差异表达基因 (differentially expressed genes, DEGs);随机选取8个上调和12个下调差异表达miRNA,对其表达及其预测的5个靶基因进行qRT-PCR验证;对DEGs以及差异表达miRNA的靶基因进行KEGG通路富集分析。【结果】从精巢和卵巢样本中(Test vs Control)分别鉴定出68个差异表达miRNA和3 991个DEGs,其中上调和下调miRNA分别为36和32个,上调和下调DEGs分别为2 033和1 958个。差异表达miRNA的qRT PCR验证结果均与芯片数据一致。KEGG通路富集分析结果显示DEGs在新陈代谢及核糖体的信号通路显著富集。对差异表达miRNA在DEGs中的可能靶基因进行预测,结果找到了4组表达趋势相反的miRNA与靶基因：分别是bmo-miR-2774a与LOC101745556;bmo-miR-92b与LOC101735954以及bmo-miR-3266与LOC733130和LOC778467;1组表达趋势一致的miRNA与靶基因：bmo-miR-3321与LOC101744895。5个靶基因的qRT-PCR验证结果与转录组测序结果一致。【结论】本研究获得了家蚕5龄幼虫精巢和卵巢转录组及miRNA芯片数据,筛选并验证了4组差异表达和1组一致表达miRNA及潜在靶基因,为探究家蚕精巢和卵巢发育差异奠定了基础。     

家蚕CRISPR/Cpf1基因编辑系统建立

自CRISPR/Cas9基因编辑系统成功应用于模式生物以来,因其快速、高效、便捷等特点,广泛应用于基因功能研究、基因治疗和基因工程等研究领域。与此同时,CRISPR/Cas系统不断在微生物界的发现也加速了新的基因编辑工具的不断涌现。CRISPR/Cpf1是第二类 (Ⅴ型) 能够编辑哺乳动物基因组的CRISPR系统,相比于CRISPR/Cas9基因编辑系统,能够利用5′T-PAM富集区增加基因组覆盖率,具有其切割位点为粘性末端和更不易同源重组修复等诸多优势。基于此,本研究构建了能够在家蚕细胞表达的3个不同来源的CRISPR/Cpf1 (AsCpf1、FnCpf1和LbCpf1) 表达载体,选择高度保守的家蚕热休克蛋白基因BmHSP60和家蚕ATP酶家族BmATAD3A基因分别设计靶标gRNA,构建gHSP60-266R和gATAD3A-346R基因编辑载体。通过T7E1酶切分析和T克隆测序,鉴定3个Cpf1基因编辑系统AsCpf1、FnCpf1和LbCpf1对靶标基因BmHSP60和BmATAD3A的编辑效率。同时,利用Western blotting分析不同基因编辑系统敲除靶基因后对其BmATAD3A和BmHSP60蛋白翻译的影响。本研究成功构建了家蚕CRISPR/Cpf1基因编辑系统,能够在家蚕细胞中有效编辑家蚕基因组,为家蚕基因功能研究、基因工程和遗传育种开发了新技术与新方法。