家蝇蛹凝集素的免疫调节作用

本实验研究了从家蝇蛹体内分离的一种凝集素,研究了其免疫调节的性质。首先将收集的家蝇蛹在缓冲液中研磨,得到粗提物,经过亲和吸附、竞争洗脱等步骤得到了纯品。电泳结果表明家蝇蛹凝集素分子量大约为55kD。通过检测巨噬细胞分泌的细胞因子IL-6、IL-12等实验,证明了家蝇蛹凝集素浓度在5μg/mL时对与巨噬细胞分泌IL-6有显著增强作用,家蝇蛹凝集素的浓度在10μg/mL时对巨噬细胞分泌IL-12有显著效果。通过小鼠脾淋巴细胞增殖试验结果可知家蝇蛹凝集素对于混合淋巴细胞增殖有促进作用。以上试验结果说明家蝇蛹凝集素免疫调节作用,为天然免疫增强剂的开发提供了一定的参考依据。     

Diuresis in the housefly (Musca domestica) and its control by neuropeptides

Diuresis was studied in vivo by measuring the loss of tritiated water. The basal rate of water loss (5 nl/min) represents respiratory and cuticular losses, whereas higher rates reflect urine output, which reaches 20 nl/min after injection of 1 microl distilled water. This response to hypervolemia involves release of a diuretic hormone(s) into the hemolymph. However, housefly diuretic peptides increased urine output to a maximum of only 7 nl/min, and higher rates may require fluid reabsorption from the hindgut to be reduced. Diuresis is partially blocked by injected anti-muscakinin antibodies, providing evidence of a hormonal function for this insect myokinin.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

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。     

Testing alternative methods for purging genetic load using the housefly (Musca domestica L.)

When a population faces long-term inbreeding, artificial selection, in principle, can enhance natural selection processes for purging the exposed genetic load. However, strong purge pressures might actually decrease fitness through the inadvertent fixation of deleterious alleles and allelic combinations. We tested lines of the housefly (Musca domestica L.) for the effectiveness of artificial selection to promote the adaptation to small population size. Specifically, replicate populations were held at average census sizes of 54 for nine generations or 30 for 14 generations while being subjected to artificial selection pressure for increased fitness in overall mating propensity (i.e., the proportion of virgin male–female pairs initiating copulation within 30 min), while also undergoing selection to create differences among lines in multivariate components of courtship performance. In the 14-generation experiment, a subset of the lines were derived from a founder-flush population (i.e., derived from three male–female pairs). In both experiments, we also maintained parallel non-selection lines to assess the potential for natural purging through serial inbreeding alone. Sub-populations derived from a stock newly derived from the wild responded to artificial selection for increased mating propensity, but only in the short-term, with eventual rebounds back to the original levels. Serial inbreeding in these lines simply reduced mating propensity. In sub-populations derived from the same base population, but 36 generations later, both artificial selection and serial inbreeding increased mating propensity, but mainly to restore the level found upon establishment in the laboratory. Founder-flush lines responded as well as the non-bottlenecked controls, so we base our major conclusions on the comparisons between fresh-caught and long-term laboratory stocks. We suggest that the effectiveness of the alternative purge protocols depended upon the amount of genetic load already exposed, such that prolonged periods of relaxed or altered selection pressures of the laboratory rendered a population more responsive to purging protocols.     

Dietary cholesterol utilization by the housefly,Musca domestica

The amount of cholesterol and the distribution of its metabolites were studied in various larval tissues of the housefly (Musca domestica), reared on a high cholesterol diet containing the radioactive cholesterol. The sterol content was also examined in adults derived from larvae reared on increasing dietary cholesterol concentration in separate batches. The results suggest that when the dietary sterol concentration was raised from 0.002 to 0.02% wet weight (an optimal concentration required for a maximal growth and development of insect), the sterol content of six-day old larvae was increased approx. 4-fold. However, a further 10-fold increase of dietary cholesterol (to 0.22% of wet weight) was associated with only a 1.5-fold increase in larval sterol content, and no increase in the sterol content of adult insects derived from such larvae was observed. This increase of sterol content of whole larvae was found to be confined to the larval cuticle and composite gut fractions and may be attributed to unabsorbed cholesterol in the gut, and to a solubilizing effect of the cuticle. These findings suggest that when the cholesterol requirement for the maximal growth of the insect has been reached, larvae are able to regulate the intake of ingested cholesterol, and no more is taken up through the gut even when a high gut concentration of cholesterol is present.The adults contained a higher percentage of esterified sterols than the larvae, approx. 25% of the sterol in females and 14% of sterol in males were esterified.     

Circadian rhythm gene regulation in the housefly Musca domestica

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.     

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.     

家蝇抗菌肽Attacin-2基因的克隆、序列分析和诱导表达

攻击素(attacin)作为昆虫抗菌肽之一, 在昆虫的先天免疫中起着重要作用。本研究通过家蝇Musca domestica EST序列筛选并结合RACE技术克隆了家蝇的Attacin-2基因(Mdatta2) cDNA序列。其全长819 bp, 包含一个726 bp的完整开放阅读框(open reading frame, ORF), 以及42 bp的5′末端非翻译区(5′-UTR)和51 bp的3′末端非翻译区(3′-UTR), 编码241个氨基酸残基, 推导的多肽N端22个氨基酸残基为信号肽序列。同源性分析表明, 其氨基酸序列与嗜凤梨果蝇Drosophila ananassae的Attacin一致性最高(46%)。以邻接法(Neighbor-Joining, NJ)构建的系统关系表明, 家蝇的Attacin-2与其他双翅目昆虫的Attacin起源于共同的祖先, 属于Attain_C超家族。应用实时荧光定量PCR的方法研究家蝇幼虫在受到外源细菌刺激时Mdatta2基因的表达, 结果显示, 在大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus分别刺激后3 h和6 h, 家蝇幼虫Mdatta2表达量出现显著上调。Mdatta2基因在家蝇幼虫体内呈诱导型表达, 表达水平随诱导时间的不同而变化, 提示Mdatta2基因可能在家蝇免疫防御过程中起着重要作用。