Persistence of antibiotic-resistant and -sensitive Proteus mirabilis strains in the digestive tract of the housefly (Musca domestica) and green bottle flies (Calliphoridae)

Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.     

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.     

The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenebiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from 5 different Turkish housefly strains. High doses of malathion (600 microg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 --> Ser substitution was found in the product of MdalphaE7 gene in all malathion resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all 5 strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion resistant strains.     

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。     

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.     

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。     

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。     

Occurrence and metabolism of prostaglandins in the housefly,Musca domestica (L.)

Prostaglandins (PGs) of the E and F series were quantified in the housefly by radioimmunoassay (RIA). Whole insects and reproductive tissues from both sexes contained PGE₍₁₊₂₎ and PDG_2α which increased in amount with age. PGF_2α levels were higher than PGE series in extracts of whole male and female insects and in ovaries. Male reproductive tissues contained higher amounts of PGE₍₁₊₂₎ than PGF_2α. Gas chromatographic-mass spectrometric (GC-MS) analyses of the products formed after injection of arachidonic acid (20:4) and eicosatrienoic acid (20:3, n-6) into male and femal insects demonstrated the conversion of 20:4 to PGF_2α and 20:3 to PGF_1α. Radiolabeled 20:4 injected into houseflies was rapidly converted to PGE₂ and PGF_2α. The catabolism of PGE₂ was more rapid than PGF_2α in males, whereas in females, PGE₂ and PGF_2α were converted to more polar products at similar rates. Radiolabeled 20:4 injected in the hemolymph was incorporated into the reproductive tracts of male insects. About 2.1% of the total radioactivity from [³H]20:4 injected into males just prior to mating was transferred to females during mating. Thus, PG are formed from 20:4 in male and female houseflies. During mating, 20:4 is transferred from males to females where it can be metabolized to PGF_2α.     

Characterization of prostaglandin synthesis in the housefly,Musca domestica (L.)

The in vitro formation of prostaglandins (PG) was examined in the housefly Musca domestica. PG synthetase activity was detected in homogenates of whole insects and in head and thorax, abdomen, ovary and male reproductive tissues. Studies to determine the sub-cellular localization of PG synthetase indicated that the microsomal fraction contained the highest activity. Products obtained from radiolabeled arachidonic acid (20:4) and 8,11,14-eicosatrienoic acid (20:3, n-6) were PGE₂ and PGE₁, respectively, with lower amounts of the PGF series also present. In microsomal preparations from whole insects and reproductive tissues from both males and females, 20:3(n-6) was 2–2.5 times more efficiently converted to PG than was 20:4. Non-steroidal anti-inflammatory drugs (NSAID) fed to houseflies did not inhibit PG production from 20:4, whereas when they were included in microsomal preparations at high levels, they inhibited PG synthesis.     

家蝇介导的水稻基因逃逸风险评估(英文)

【目的】对转基因作物进行生态风险评估是大面积种植前的一个必要步骤,水稻Oryza sativa访花昆虫有上百种,包括家蝇Musca domestica。本研究旨在明确访花昆虫家蝇介导转基因水稻外源基因逃逸的风险。【方法】2010年,我们使用转基因水稻B1, B6和G8-7作为父本(花粉供体),用同源非转基因水稻Jiazao 935和Wuyunjing 7作为母本（花粉受体）,并用家蝇作为授粉昆虫,在浙江大学华家池和长兴试验基地开展了田间种植试验,对收割的后代水稻种子进行室内种植培养,对种苗用潮霉素B和草甘膦处理进行转基因杂交种检测,对存活植株进行潮霉素和草甘膦抗性基因PCR检测,测试家蝇介导的转基因水稻外源基因逃逸频率。【结果】对浙江两个测试基地3个转基因水稻品种共计超过216 500粒后代水稻种子进行的检测及结果表明,在毗邻区域杂交种少,家蝇授粉区和无家蝇授粉区转基因水稻外源基因向非转基因水稻逃逸频率均较低(0~0.64%)。【结论】家蝇介导的转基因水稻外源基因逃逸频率较低,家蝇没有增加转基因水稻外源基因逃逸的风险。