家蚕保存系统红色卵的遗传分析

用遗传背景清楚的家蚕Bombyx mori红卵（re）、白卵(w-2、pe)、第4褐卵（b-4）的标志基因系统和正常型黑卵系统与我国家蚕基因库保存的20个红色卵系统杂交,进行顺反测验,分析了它们的卵色支配基因及遗传规律。结果发现：①在03-310系统中存在家蚕卵色新突变pink egg,与红卵re 等位,基因符号为re^p,表型特征为：卵淡红色,成虫蛾眼也为淡红色;②6个系统为红卵（re）的纯合系统,还有５个系统除具有re／re基因型外,还具有支配白色卵或浅红色或橙红色卵的突变基因;③２个系统为第4褐卵（b4）的纯合系统; ④6个系统的红褐色卵为母性影响遗传;⑤发现家蚕卵色基因b-4和r-e的互补关系,b-4/b-4 re/re基因型表现为新的卵色——橙黄色。     

Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm,Bombyx mori

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth‐instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.     

Proteome analysis on lethal effect of <Emphasis Type="Italic">l</Emphasis><Subscript>2</Subscript> in the sex-linked balanced lethal strains of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex-linked balanced lethal (SLBL) strains of silkworm serve as an effective system for sex-control in silkworm. To gain comprehensive insight into the effect of one sex-linked balanced lethal gene l ₂, comparative proteomic analysis was carried out between the survival embryos ( W ^{+ l₁} Z^{l₁ + l₂} )\left( {W^{ + l_1 } Z^{l_1 + l_2 } } \right) and lethal embryos ( W ^{+ l₁} Z ^{+ l₁ l₂} )\left( {W^{ + l_1 } Z^{ + l_1 l_2 } } \right) before the lethal stage. The lethal stage of l ₂ was confirmed by observing the typical dead embryo morphology. The two genotype embryos before lethal stage were distinguished using polymorphic simple sequence repeats (SSR) markers closely linked to l ₂ on the sex chromosome. Finally, 11 differentially expressed protein spots were successfully identified by MALDI-TOF/TOF mass spectrometry (MS). Among them, only 1 protein identified as heat shock protein 20.4 (HSP20.4) was up-regulated in the lethal embryos, while the other 10 were down-regulated. The up-regulation of HSP20.4 suggests that there may be abnormal polypeptides produced in the lethal embryos. The gene ontology (GO) annotation indicated those down-regulated proteins are involved in important biological processes including embryo development, nucleoside metabolism, tRNA splicing, translation and protein folding. The biological pathway analysis showed that those down-regulated proteins are mainly involved in spindle assemblage and morphogenesis. Based on our results, we suggest that the l ₂ may be the mutant expressing abnormal polypeptides. Its expression has a negative effect on mitosis and morphogenesis processes. The death of the embryos may be caused by the accumulation of abnormal polypeptides and the handicap of cell proliferation and morphogenesis.     

Short communication. The effect of temperature, food concentration and female size on the egg production of the planktonic copepod Acartia bifilosa

Egg production of a brackish water calanoid copepod Acartia bifilosa was measured in the laboratory in different chlorophyll (Chl) a concentrations (0-24 mgr; l^-1) and temperatures 4-24C), and the cephalothorax length and carbon content of females were determined. Egg production was positively correlated both with Chl a concentration and with temperature; highest egg production was obtained with 14-20 g Chl a l^-1 and at 13-18°C. There was also a significant positive correlation between egg production and female length-specific carbon content (g C m^-1). However, no correlation was observed between egg production and cephalothorax length of females. Female carbon content changed during the 3 day experiments; carbon content was positively related to Chl a concentration and negatively related to temperature. We conclude that food availability (Chl a concentration), rather than temperature, limits the egg production of A.bifilosa in the present study area in the northern Baltic Sea. Further, both food concentration and temperature affect egg production not only through the direct effect on the numbers of eggs produced per female, but also through their effect on female carbon content.      

一种新的家蚕体形突变体——短体蚕（Sq）的遗传分析与基因定位

作为重要经济昆虫和鳞翅目昆虫模式的家蚕Bombyx mori,其突变体是生理学、遗传学、功能基因组学等研究的宝贵资源。作者在家蚕资源保存和遗传分析中发现一种新的体形突变体——短体蚕（Squab,Sq）,其特征是：杂合体（Sq /+）成活,蚕体长只有正常型的约4/5,腹中部略肥大,胸部稍狭小;纯合体（Sq / Sq）胚胎期致死。遗传分析结果表明该突变为显性遗传。通过与各染色体标记基因进行连锁分析,发现突变基因Sq在家蚕第14染色体上;通过与同一染色体上的标记基因青熟油蚕基因（oa）、不洁蚕基因（Di）进行三点测验,将Sq定位在家蚕连锁图谱第14连锁群的34.6 cM位点,表示为Sq（14-34.6）。本研究结果为深入研究和利用该突变体奠定了重要基础。     

A novel system for separation and purification of egg pigments from the nondiapause red-egg mutant Re-nd in the silkworm,Bombyx mori

Re-nd, which was induced from the wild-type C108 by the chemical mutagen N-methane-N-methylnitrourea, is a nondiapause red-egg mutant of silkworm Bombyx mori. The special significance of the Re-nd mutant is that it is an independent dominant mutant. The aim of this study was to establish the type of pigment responsible for the red coloration in the Re-nd mutant eggs in silkworm. We compared the eggs of Re-nd mutants with those of the other B. mori egg color strains and confirmed that the Re-nd mutant is the only strain with red color and red pigment granules in nondiapause, showing this mutant belongs to the pigmentation in the serosa. We speculated that the red substance, which contributed to the bright red pigmentation for nondiapause eggs of the Re-nd mutant, could potentially be a novel pigment according to its solubility, optimum absorption peak, and oxidation–reduction reaction. Moreover, we have successfully constituted the system for enrichment, extraction, and purification of the red substance responsible for the Re-nd mutant, providing a new method for the separation and purification of other known and unknown pigments or substances.     

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Background  

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3^rd day of fifth instar, the 6^th day of fifth instar, the 3^rd day of pupation, the 8^th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.     

Genetic investigation of contributions of embryo and endosperm genes to malt Kolbach index, alpha-amylase activity and wort nitrogen content in barley

 A genetic model is proposed for the analysis of embryo and endosperm effects as well as GE interaction effects. An investigation of three malting quality traits in grains of seven parents and their F₂s was undertaken in a half-diallel cross of barley (Hordeum distichum L.) over 2 years. The results indicated that the malt Kolbach index (KI), alpha-amylase activity (αAA) and wort soluble nitrogen (Wort-N) are controlled by both embryo genetic effects and endosperm genetic effects. Variance of the endosperm additive effects was obviously larger than that of the embryo additive effects. In the contribution of the embryo genetic effects to variation in malt αAA and Wort-N, the dominance effects were considerably larger than the additive effects. The endosperm dominance effects constituted a major part of the total genetic effect on the KI. Significant endosperm GE interactions were also detected in the malt traits concerned. Endosperm general heritability (h ² _e ) tended to be larger than interaction heritability (h ² _oE or h ² _eE ) for all the traits. Endosperm heterosis was observed to be significantly positive for αAA but negative for Wort-N in the F₂ seed generation. Prediction of main gene effects for seven parents showed that ‘Ganmu 2’ and ‘Supi1’ were suitable parental varieties for malt αAA and Wort-N improvement. Our genetic model for malting quality traits and its application in breeding are discussed. Received: 5 August 1997 / Accepted: 11 September 1997     

Is the egg size determining gene,Esd, on the W chromosome identical with the sex-linked giant egg gene,Ge, in the silkworm?

The presence of the egg size-determining (Esd) gene, which acts as a quantitative gene, on the W chromosome of the silkworm was revealed in a previous study by using two types of triploid females, ZZW and ZWW, (Kawamura, Genetica 76: 195–201). The females with the sex-linked giant egg (Ge) gene deposit eggs as large as those laid by tetraploids. If the Ge mutant is induced by translocation of a fragment of the W carrying Esd onto the Z by chance, the egg size increase in the Ge strain and in tetraploids may be easily explained by the double dose of Esd. The measurement of the length of the Z-W bivalent in oocytes showed that the Z of the Ge strain was much longer than that of the other strains which do not carry the Ge gene. The result suggests that the Ge gene is identical with the Esd on the W chromosome of the silkworm.     

A laboratory assessment of the potential molluscicidal activity of some Nigerian plant species used as anthelmintics

Laboratory studies were conducted to determine the potential molluscicidal activity of five Nigerian plant species used in alternative medicine practice as anthelmintics. Laboratory-bred adult Biomphalaria pfeifferi, the snail host of Schistosoma mansoni in Nigeria, and their viable 0- to 24-hour-old egg masses were separately exposed for 24 hours, in replicated bioassays, to 7.81–4 000 mg l^?1 concentrations of crude ethanolic extracts of the leaves and roots of Annona senegalensis, Anogeissus leiocarpus, Crotalaria retusa, Dalbergia sissoo and Vernonia amygdalina. Fruits and stem bark of D. sissoo were also investigated. The 24-hour LC50 and LC90 values of extracts for target organisms were calculated using probit analysis. Susceptibility of target organisms to extracts varied with the plant species and morphological part. Egg masses and adult snails were most susceptible to D. sissoo fruit extract (LC90 values of 89.29 and 74.33 mg l^?1, respectively) and least susceptible to V. amygdalina leaf extract (LC90 values of 11 864.09 and 6 241.61 mg l^?1, respectively). Concentration-dependent behavioural responses and embryo deaths at the gastrula/exogastrula and/or prehatch snail stages of development were observed in exposed snails and egg masses, respectively. Tested extracts of A. senegalensis, A. leiocarpus, C. retusa and V. amygdalina did not show appreciable toxicities to B. pfeifferi egg masses or adults. For demonstrating significant molluscicidal activity (LC90 < 100 mg l^?1) and toxicities to egg masses, we recommend further studies on the ethanolic extracts of D. sissoo fruits and roots.     

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

Developmental response of the beneficial predator Podisus maculiventris to change in dietary ascorbic acid concentration

We report here the effects of a range of ascorbic acid concentrations (0.07, 0.3, 3.0, and 30.0 g l^?1) in artificial diets on growth rates, adult weights, fecundity, and survival over two generations of the predatory stink bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Overall, a dietary level of 3.0 g l^?1 gave the shortest developmental times over two generations. The likelihood of egg hatch at one ascorbic acid concentration compared to another concentration suggested that egg hatch increased as the concentration of ascorbic acid increased from 0.07 to 3.0 g l^?1 and then declined from 3.0 to 30.0 g l^?1. The combination of the maximum egg oviposition at 0.3 and 3.0 g l^?1, egg hatch at 3.0 g l^?1, and survival at 0.07 and 0.3 g l^?1 suggests an overall superior performance at a concentration between 0.3 and 3.0 g l^?1. Depletion of ascorbic acid below 3.0 g l^?1 or addition of ascorbic acid above 3.0 g l^?1 lowered the likelihood of egg hatch, which became more pronounced in the second generation. This is consistent with previously published information for phytophagous insects.     

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm “Ming” lethal egg mutant

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-e^m) lose water and become concaved around 1 h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e^m mutant to 360 kb on chromosome 10 using 2287 F₂ individuals. Using expression analysis and RNA interference, the best l-e^m candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~ 1.9 kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a > 100 kb DNA fragment in the l-e^m mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e^m mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e^m mutant. These results indicate that BmEP80 is responsible for the l-e^m mutation.     

家蚕动力蛋白轻链8基因克隆及其对重力的响应

克隆家蚕动力蛋白轻链8(dynein light chain 8, Dlc8)基因开放阅读框架并对其序列进行分析．探讨Dlc8基因在家蚕胚胎、头、丝腺、中肠、皮肤、血液、脂肪和马氏管等组织中的分布．在细胞水平,应用RT-PCR和实时定量RT-PCR方法分析大梯度强磁场(large gradient high magnetic field, LGHMF)重力模拟环境(0 g、1 g、2 g)对家蚕Dlc8基因表达的影响．在整体水平,应用实时定量RT-PCR方法分析Dlc8基因在家蚕胚胎反转期和整个胚胎期对LGHMF模拟失重环境的响应．克隆的家蚕Dlc8基因开放阅读框架长度为270 bp,编码89个氨基酸．家蚕Dlc8基因推导的氨基酸序列与拟南芥(Arabidopsis thaliana)、果蝇(Drosophila melanogaster)、线虫(Caenorhabditis elegans)、热带爪蟾(Xenopus tropicalis)、小鼠(Mus musculus)、人类(Homo sapiens)等6个物种Dlc8基因的氨基酸序列同源性分别为67%、96%、91%、95%、92%、92%．信号肽分析结果显示,该蛋白质为非分泌蛋白,不存在糖基磷脂酰肌醇锚定位点．家蚕Dlc8分子质量与等电点分别为10.34 ku和6.81．Dlc8基因在家蚕的胚胎、头、丝腺、中肠、皮肤、血液、脂肪、马氏管中稳定表达．在细胞水平,家蚕Dlc8基因表达对重力变化较敏感,对磁场变化不敏感．在整体水平,Dlc8基因在家蚕胚胎发育的不同时期对重力的响应不同．整个胚胎发育期Dlc8基因在模拟失重条件下表达量与对照组接近．家蚕Dlc8基因可以作为重力生物学效应研究的分子靶标．该研究为深入探讨家蚕Dlc8基因重力生物学效应机制奠定了基础．