Regulation and metamorphosis of the abdominal histoblasts ofDrosophila melanogaster

Summary The development of the adult abdomen ofDrosophila melanogaster was analyzed by histology, microcautery, and genetic strategies. Eight nests of diploid histoblasts were identified in the newly hatched larva among the polytene epidermal cells of each abdominal segment: pairs of anterior dorsal, posterior dorsal, and ventral histoblast nests and a pair of spiracular anlagen. The histoblasts do not divide during larval life but begin dividing rapidly 3 h after pupariation, doubling every 3.6 h. Initially they remain confined to their original area, but 15 h after pupariation the nests enlarge, and histoblasts replace adjacent epidermis cell by cell. The histoblasts cover half the abdomen by 28 h after pupariation and the rest by 36 h. Polytene epidermal cells of the intersegmental margin are replaced last. Cautery of the anterior dorsal nest caused deletion of the whole corresponding hemitergite, whereas cautery of the posterior dorsal nest caused the deletion of the macrochaetae of the posterior of the hemitergite. Cautery of the ventral nest deleted the hemisternite and the pleura, whereas cautery of the spiracular anlagen deleted the spiracle. Results of cautery also revealed that no macrochaetae formed on the tergite in the absence of adjacent microchaetae. Clonal analysis revealed that there were no clonal restrictions within a hemitergite at pupariation. Cautery of polytene epidermal cells other than those of the intersegmental margin failed to affect tergite development. However, cautery of polytene epidermal cells of the intersegmental margin adjacent to either dorsal histoblast nest caused mirror-image duplications of the anterior or posterior of the hemitergite in 10% of the hemitergites. Forty percent of the damaged presumptive hemitergites formed complete hemitergites, indicating extensive pattern regulation and regeneration. Pattern duplication and regeneration were accounted for in terms of intercalation and a model of epimorphic pattern regulation (French et al., 1976). Histoblasts in adjacent segments normally develop independently, but if they are enabled to interact by deleting the polytene epidermal cells of the intersegmental margin, they undergo intercalation which results in duplication or regeneration. The possible role of the intersegmental margin cells of insects in development was analyzed.     

蝇蛆蛋白粉的营养评价

对以盐提-酸沉淀法获得的蝇阻MuscadomesticavicinaWacquart蛋白粉进行营养评价,其蛋白质含量为73.07％,脂肪为23.01％,灰分为1．83％,提取的蛋白质含量比蛆粉高18.0％,脂肪高11．5％,灰分低9．6％;氨基酸中除丙氨酸含量低于蛆粉外,其余氨基酸含量均高于蛆粉,必需氨基酸配比合理,EAA值为41.6%。动物实验采取AOAC方法,测得蛋白质效价（PER)为2．67±0．34,表观消化率（AD)为84.40±2．5％,生物价（BV)为78.68±5．71％,净蛋白利用率（NPU)为666.29±4.19％,蝇蛆蛋白粉被动物摄取后在动物体内的吸收程度比蛆粉及酪蛋白高。实验结果表明：蝇蛆蛋白粉是1种优质蛋白质。     

家蝇幼虫体内一种抗菌蛋白的分离纯化

30%H2O2诱导家蝇幼虫90min,继续饲喂24h后利用硫酸铵分级盐析、Sephadex G-25 和Sephadex G-75两步凝胶过滤、CM-Sepharose Fast Flow弱阳离子交换的纯化方法,得到一种电泳纯的抗菌蛋白,经过VDS凝胶扫描得到其分子量为28kDa,命名为AP28。抑菌活性分析表明,AP28对实验中涉及的大多数革兰氏阳性菌和阴性菌都有明显的抑制作用。     

Fine structural alterations with age in the fat body of the adult male housefly,Musca domestica

Summary The fat body of the adult housefly is composed of two types of cells, the lipid-and glycogen-rich fat body cells and the oenocytes. A comparison of the fine structure of the abdominal fat body in 4-day old and 31–35 day old male houseflies indicated an increase in lipid and a decrease in glycogen content in the fat body cells of old flies. Oenocytes of old flies exhibit deteriorative alterations with an accumulation of secondary lysosomes. Both fat body cells and oenocytes in senile flies are ingested by hemocytes.Supported by a grant from the National Science Foundation.     

Lanthanum and freeze fracture studies on the retinular cell junction in the compound eye of the housefly

Summary The retinular (R) cell junction between adjacent photoreceptor cells in the house-fly ommatidium was characterized by freeze fracture, thin section and tracer (lanthanum) studies. Focal tight junctions occur between cells, and some P face ridge-E face groove correspondences are present in this intramembranal area. When colloidal lanthanum was introduced into the extracellular space (ECS) of the peripheral retina of the housefly, this electrondense tracer moved from the ECS (extra-ommatidial space), through the R-cell junctions and belt desmosomes, into the ommatidial cavity (OC = intrarhabdomal space) of each ommatidium. In the OC, lanthanum outlined a meshwork structure that pervaded this space. The evidence of this tracer movement suggests that there may be ionic continuity between the traditional ECS and the fluid bathing the individual rhabdomeres. The volume of the OC is calculated and we suggest that this space is part of the ECS. The functional implications of this postulate are considered in the light of: (1) the different functions of the peripheral and central cells; (2) the dissimilarity of rhabdomal membrane surface facing the OC compared to the unmodified plasma membrane of the photoreceptor cell facing the extra-ommatidial cavity; (3) the permeability properties of the R cell junction; and (4) the total ECS containing an ion store capable of sustaining current for the generator potential.We gratefully acknowledge support from the N.I.H., National Eye Institute, EYO 1686 and from the College of Agricultural and Life Sciences, Hatch Project 2100. We thank Dr. Philippa Claude (UW Primate Research Center, Madison) for training in freeze fracture technique. This part of the work was supported by Grant RR00167 from the N.I.H. to the Primate Research Center. Dr. Robert Goy, Director of the Primate Research Center is acknowledged for his kind permission to use the Center's freeze fracture apparatus. Professor Stanley D. Beck, Department of Entomology, UW, Madison provided valuable advice for which we thank him heartily     

家蝇幼虫集约化生产的初步研究

对家蝇幼虫在人工条件下的集约化饲养进行了研究．结果表明,在２４０ｍ２场地（日生产单元６０ｍ２）上得到鲜蛆日产量最高超过２００ｋｇ,平均约１５０ｋｇ;蝇蛆自卵孵出的第２天是蝇蛆生长的最关键阶段,此时要严格控制环境条件和饲料质量;同日龄的成蝇群体开始产卵后,约３ｄ便进入产卵高峰期并维持３～４ｄ;雌蝇一生产卵１０～１４次,每产１００～２００粒,最多９００多粒;蝇卵约１ｄ即孵化成幼蛆,由卵到成品蛆的时间需５～６ｄ;蝇蛆体重的增长过程符合Ｌｏｇｉｓｔｉｃ曲线;蛆料比０．４～０．５,产投比２．１～３．３;试验条件下,家蝇经过一个世代后个体数的实际倍增为理论倍增的２５．５％．利用农业生产中的各种副产品和废弃物进行蝇蛆的集约化生产,以获得高质量的动物蛋白和精饲料是可行的途径     

蝇蛆几丁低聚糖咀嚼片的调节血脂作用

研究和评价了蝇蛆几丁低聚糖咀嚼片的调节血脂作用及其安全性。将50只实验动物（大鼠）随机分为普通饲料对照组、高脂饲料对照组和3个实验组,实验组分别喂以不同剂量蝇蛆几丁低聚糖咀嚼片和高脂饲料。饲养28天后,分别测定大鼠血清总胆固醇（total cholesterol, TC）、血清总甘油三酯（triglyceride, TG）、血清高密度脂蛋白胆固醇（high density lipoprotein cholesterol, HDLC）的含量。用Horn's法对蝇蛆几丁低聚糖咀嚼片的急性毒性进行研究,连续观察7天,记录各组动物的中毒反应情况和死亡只数,计算咀嚼片对小鼠的半数致死剂量。同时对几丁低聚糖咀嚼片作了调节血脂的动物试验,研究结果表明,蝇蛆几丁低聚糖咀嚼片对大鼠血清总胆固醇、总甘油三酯有明显降低和对血清高密度脂蛋白胆固醇有明显稳定作用,具有辅助降血脂作用;对咀嚼片进行急性毒性实验表明,蝇蛆几丁低聚糖咀嚼片对小鼠的半数致死剂量大于10 g/kg·bw,提示其基本无毒。
     

The perineurium of the adult housefly: Ultrastructure and permeability to lanthanum

Summary The ultrastructure of the perineurial cells of Musca overlying the first optic neuropile was examined by transmission electron microscopy. These cells are somewhat similar to those of other insects but cytoplasmic flanges seem to be absent, and mitochondria are relatively large and sinuous. The intercellular channel system on the lateral border of the cells is relatively spacious and highly meandering. Perineurial cells are joined by septate, gap, and tight junctions, hemidesmosomes, and desmosomes. Tight and septate junctions bond perineurial cells and glial cells. These data are evaluated on the basis of tracer studies with lanthanum. This material penetrates the extracellular space between perineurium and underlying glial and nerve cells, between epithelial glial cells and retinular axon terminals (capitate projections), and between the - fiber pair in the optic cartridge (gnarls). If no damage occurs to the perineurial cells during tissue preparation, this passage of lanthanum to neuronal surfaces indicates that the blood brain barrier is incomplete in this restricted area. Supportive evidence for such permeance is based on electrophysiological data, considerations of membrane specializations in the optic neuropile, and Na⁺/K⁺ ratios of dipteran hemolymph.We gratefully acknowledge support from the N.I.H., National Eye Institute, EYO 1686 and from the College of Agricultural and Life Sciences, Hatch Project 2100. Richard L. St. Marie and Professor Stanley D. Beck, Department of Entomology, UW, Madison read early drafts of this paper and provided constructive comments     

Kinetic characteristics of native gamma-glutamylcysteine ligase in the aging housefly, Musca domestica L

The catalytic activity of gamma-glutamylcysteine ligase (gamma-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes-Woolf analyses ([S]/v vs [S]) indicated that gamma-GCL had significantly higher affinities for its substrates in the young than in the old flies. The K(m) values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM; for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of gamma-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native gamma-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies.     

家蝇防御素在大肠杆菌中的表达、纯化与抗体制备

家蝇防御素是从家蝇中克隆得到的1种抗菌肽。为了进一步研究家蝇防御素的功能和制备特异性抗体,采用大肠杆菌表达外源蛋白的方法, 进行了家蝇防御素原核表达的研究。根据克隆到的家蝇防御素基因(Mdde) 的cDNA序列, 设计特异性引物, PCR 扩增成熟肽的cDNA片段, 将成熟肽序列重组到表达载体pGEX 4T 1中, 构建m Mdde/pGEX 4T 1重组表达载体, 在大肠杆菌BL21 中诱导表达, 重组表达的融合蛋白GST Mdde占菌体总蛋白的33 4%。纯化得到GST Mdde后, 再用凝血酶将其从特定位点切开, 得到表达的m Mdde。液体抑菌实验结果初步表明, 表达的融合蛋白GST Mdde对细菌生长有一定的抑制作用。利用纯化的GST Mdde融合蛋白, 制备了抗血清。     

A complex glutathione transferase gene family in the housefly Musca domestica

In most metazoans, the glutathione S-transferases (GST) are encoded by gene families, and are used to detoxify xenobiotics. We describe the structure of genomic loci coding for the GSTs in the housefly that have been implicated, by both genetic and biochemical means, in mediating insecticide resistance. In earlier work, we showed that one of the theta-class enzymes, MdGST-3, is overproduced in resistant flies and degrades certain insecticides. We used a fragment from a cDNA clone of MdGST-3 as a probe to screen a housefly genomic DNA bank in phage λ. This probe detected multiple gst loci. Genes for GSTs were found in five different, nonoverlapping λ clones, three of which carry multiple, closely linked gsts. Multiple genes for both MdGST-3 and MdGST-4 were found; some of which have introns in their 5′ untranslated regions. In adults, the only MdGST-3 enzymes that are expressed are encoded by the intron-free genes. A new theta-class GST (called MdGST-5) was also discovered. Fusion genes comprising 5′ MdGST-3 sequences and either MdGST-4 or MdGST-5 sequences in their 3′ halves were encountered at three separate loci. The genes described here are found in both the ancestral sensitive strain and the insecticide-resistant strains. Received: 28 June 1996 / Accepted: 23 April 1997     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Correlation of housefly sex pheromone production with ovarian development

Pheromone production in the housefly was monitored during oögenesis and in ovariectomized insects by gas-liquid chromatography (GLC) and radio-GLC. The presence of vitellogenic ovaries was required for the initiation of (Z)-9-tricosene (muscalure), (Z)-9,10-epoxytricosane and (Z)-14-tricosen-10-one synthesis. Methylalkane synthesis was enhanced by developing ovaries. Insects ovariectomized within 12 hr after emergence produced no detectable amounts of (Z)-9-tricosene, C₂₃ epoxide nor C₂₃ ketone and synthesized less methylalkanes than the controls. This effect was reversed by ovary implants. When flies were ovariectomized after oviposition, synthesis of (Z)-9-tricosene, C₂₃ epoxide and C₂₃ ketone continued. Thus, initiation of the synthesis of these C₂₃ pheromone compounds required a vitellogenic ovary, but the ovary was not required to maintain synthesis.     

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains.Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains.Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.     

Negative growth control of mitotically active imaginal cells (histoblasts) of the abdominal epidermis during metamorphosis of the housefly

On the ventral side of each pupal abdominal segment of the housefly, there is a pair of histoblast nests, each containing about 600 diploid cells. These cells, during adult development, divide, replace intervening polytene larval epidermal cells (LEC), and form both the median sternite and the surrounding pleura of the adult segment. Since the histoblast nests and the LEC form a contiguous layer, we examined the role of these two types of cells in regulating the mitotic potential of the histoblasts during development of the median sternite. Two experimental approaches were used: deletion of one of the nests by thermocautery; and by disturbance of the continuity of the monolayered epidermis by thermocautery of, or topical application of heptanol on, the midventral LEC. Ablation of one of the contralateral nests resulted in a mirror image duplication of the hemisternite and pleura by the surviving nest. Disturbance of the continuity of the LEC produced mirror image duplication of the hemisternal pattern by each of the contralateral nests. From these results, we propose that the contralateral ventral nests mutually downregulate their mitotic potential by secreting regulatory factor(s) to produce the normal median sternite pattern and surrounding pleura. We also suggest that these chemicals act in a paracrine fashion, possibly through gap junctions in the LEC. © 1994 Wiley-Liss, Inc.     

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Received: 22 November 1995 / Accepted: 23 February 1996     

不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响

利用不同条件提取诱导过的家蝇Musca domestica 3龄幼虫的总蛋白,并用平板扩散法测定所得蛋白粗提液的抗菌活性。结果表明,收集幼虫时的处死温度、沸水浴时间及提取液的pH值均对家蝇幼虫粗提液的抗菌活性有明显的影响。     

Pattern regulation in the ventral histoblasts of the housefly: induction of sternal pattern abnormalities by mechanical wounding of larval epidermal cells

In higher Diptera, two nests of diploid cells called the ventral histoblasts, located one on either side of each abdominal segment among the polytene larval epidermal cells, give rise to the sternite and its surrounding pleura. During metamorphosis of the insect, these two groups of cells migrate and meet with each other in the midventral region of the developing adult. The cuticular pattern elements and pigmentation in the fifth sternite of the male housefly, when compared to those of other segments as well as the tergites of both sexes, are quite distinct. The above-mentioned features, coupled with the smaller number and predictable occurrence of one of the pattern elements in this sternite, viz, the primary forceps, help one to determine the developmental potential of the histoblast nest and the regulation of its potential which occur at the time of fusion of the two contralateral nests of this segment. A simple operation of slitting the larval epidermal cells (LEC) in a hemisegment in the vicinity of the histoblast nest or extirpation or rotation of a small rectangular piece of LEC between the ventral nest and the midventral line produced pattern abnormalities including mirror image duplication in the hemisternite. An analysis of these pattern abnormalities in the different segments and, in particular, in the fifth segment provides a dynamic picture of the formation of the median sternite. Further, these abnormalities indicate the significance of the presence of the intervening pleural cells between the confronting hemisternites under experimental conditions. Thus, each of the fifth ventral nests has the developmental potential to form more than half of the final sternite pattern. Possible mechanisms for the formation of the normal median sternite during metamorphosis and for the formation of duplicated hemisternites and their fusion products under experimental conditions are discussed in light of current models of pattern regulation.