研究: 甲醛对果蝇寿命及其在应激条件下耐受性的影响

虽然甲醛对人及动物多种器官的毒性作用已经有了大量研究,但不同浓度甲醛影响动物寿命的研究还极其少见.本文用黑腹果蝇作为模式生物,在食物中添加不同浓度的甲醛,观察果蝇寿命及其在应激条件下耐受性的变化.实验结果显示,雌性果蝇的寿命表现出对甲醛浓度的依赖,0.037%甲醛可以极显著地延长雌性果蝇的寿命,较高浓度甲醛(≥0.185%)可以极显著地缩短雌性果蝇与雄性果蝇的寿命.0.037%甲醛还可以极显著地增强雌性和雄性果蝇对饥饿以及高温的耐受性,但减弱其对活性氧的耐受性.这些结果有助于从新的途径研究果蝇寿命及其在应激条件下耐受性的分子机制.     

甲醛对果蝇寿命及其在应激条件下耐受性的影响

虽然甲醛对人及动物多种器官的毒性作用已经有了大量研究,但不同浓度甲醛影响动物寿命的研究还极其少见.本文用黑腹果蝇作为模式生物,在食物中添加不同浓度的甲醛,观察果蝇寿命及其在应激条件下耐受性的变化.实验结果显示,雌性果蝇的寿命表现出对甲醛浓度的依赖,0.037%甲醛可以极显著地延长雌性果蝇的寿命,较高浓度甲醛(≥0.185%)可以极显著地缩短雌性果蝇与雄性果蝇的寿命.0.037%甲醛还可以极显著地增强雌性和雄性果蝇对饥饿以及高温的耐受性,但减弱其对活性氧的耐受性.这些结果有助于从新的途径研究果蝇寿命及其在应激条件下耐受性的分子机制.     

黑腹果蝇Mst84Db敲降引起的父本缺陷类似Wolbachia诱导的细胞质不亲和

【目的】Wolbachia 是广泛存在于昆虫体内的一类通过母系传递的共生菌,能够通过多种方式影响宿主的生殖。细胞质不亲和（CI）是Wolbachia 引起的最普遍的一种表型,即感染Wolbachia的雄性和未感染的雌性宿主交配后,胚胎发育停滞于早期阶段。但目前有关CI的分子机理还不清楚。本研究组前期实验表明,Wolbachia感染引起黑腹果蝇Drosophila melanogaster 3龄幼虫精巢中Mst84Db基因的表达显著下调。本研究的目的是进一步研究Mst84Db与CI的关系。【方法】我们体外合成了Mst84Db的双链RNA（dsRNA）,注射雄性果蝇,将注射过的雄果蝇与野生雌果蝇交配,检测其繁殖力。基因表达采用定量RT-PCR方法进行检测。胚胎表型采用DAPI染色进行分析。【结果】注射dsRNA 24 h后,Mst84Db基因的表达水平发生显著下调。注射后72 h,基因表达下调幅度最大。与对照组相比,基因敲降后的雄蝇繁殖能力显著下降,与雌果蝇交配后胚胎孵化率显著低于对照组,这与Wolbachia诱导的CI现象类似。未孵化胚胎的表皮上没有体节出现,说明胚胎停滞于发育的早期。部分胚胎细胞核分裂不同步,且有染色质间桥出现,这也与CI胚胎中的细胞学表型一致。【结论】Wolbachia感染可能抑制果蝇精子发生过程中Mst84Db基因的表达,从而使精子失去正常功能,最终导致与雌性果蝇交配后,胚胎发育停滞,并最终死亡。Mst84Db基因在雄性果蝇中表达下调可能是产生CI的重要原因之一。     

紫外辐射对野生型、黑檀体和黄体果蝇寿命、繁殖力、求爱行为及生化指标的影响

黑色素在紫外辐射（UVR）保护方面有重要作用．所以,不同体色果蝇（Drosophila malenogaster）体内色素和抗氧化能力的差异可能影响它们对UVR的敏感性．实验通过测定遭受UVR处理的野生型（w,灰色）、黑檀体（e,黑色）和黄体果蝇（y,黄色）的寿命、繁殖力和求爱行为,对三品系果蝇的UVR敏感性进行研究．UVR主要通过诱发自由基来对机体造成氧化损伤,所以,本实验通过测定果蝇体内自由基、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量来对果蝇抗氧化能力和氧化损伤状态进行分析．结果发现,w无论是在对照组还是在紫外线处理组均具有最高的寿命和繁殖力;e和y在对照组的寿命和繁殖力差异不显著,但在紫外线处理组e的这两项指标均显著高于y（P〈0．0001）．另外,本实验发现紫外辐射对果蝇的求爱行为也有不利影响．利用电子顺磁共振谱（EPR）对辐射诱发果蝇体内O水平的检测结果显示,5min紫外辐射处理的确可诱使果蝇体内自由基水平升高,且这种变化在y体内尤为明显．对SOD活性的分析发现,紫外辐射处理并不影响果蝇体内的SOD活性,因此我们将对照组与紫外线处理组的数据合并,结果发现w体内SOD活性最高,且与y的差异达显著（P〈0．05）;e和y体内SOD活性无显著差异（P〉0．05）．对MDA分析的结果显示,随UVR辐射时间的延长,果蝇体内MDA含量显著升高（P=0．0495）．以上结果说明,紫外辐射对果蝇寿命、繁殖力以及求爱行为方面的不利影响可能与其诱发的自由基氧化损伤有关．实验发现w对紫外辐射的抵抗力最强,这可能与其在自然条件下具有明显高的寿命、繁殖力有关,而且w体内高水平的SOD也可能是一个原因．与e相比,y对UVR更敏感．但作为体内重要抗氧化酶之一的SOD,并不能对现在的结果做出解释．本研究推测e和y表     

两种虫草相关真菌分级醇沉胞外多糖对果蝇寿命及SOD和MDA的影响

目的研究多糖对果蝇寿命及超氧化物歧化酶(SOD)和丙二醛(MDA)的影响。方法通过将不同浓度乙醇分级沉淀得到的两种虫草相关真菌蝙蝠蛾拟青霉和地生枝顶孢霉胞外多糖分别添加到果蝇饲料中,研究不同多糖组分对果蝇寿命及其体内SOD和MDA含量的影响。结果蝙蝠蛾拟青霉胞外多糖组分P60能显著延长雌雄果蝇的平均寿命,分别达17.04%和18.36%,显著提高雌雄果蝇体内SOD活力(28.02%和26.85%),MDA含量分别降低了50.67%和54.30%;地生枝顶孢霉胞外多糖组分G50显著延长雌雄果蝇的平均寿命,分别达到15.62%和15.96%,显著提高雌雄果蝇体内SOD活力,分别为42.55%和26.74%,明显降低MDA含量,分别为47.30%和49.00%。结论多糖组分P60和G50能显著延长果蝇寿命,提高其SOD活力和降低MDA含量。     

蛹虫草多糖对果蝇寿命影响的研究

研究了蛹虫草多糖对果蝇寿命的影响.实验结果表明:蛹虫草多糖可明显延长果蝇的最高寿命、平均寿命及半数死亡时间,具有显著的抗衰延寿作用.其中,浓度为0.15%的蛹虫草多糖可使雌性果蝇的最高寿命延长率达21.77%;使雌雄果蝇的平均寿命延长率分别达34.35%和11.24%;使雌雄果蝇的半数死亡时间延长率分别为61.76%和23%.但是蛹虫草多糖对果蝇的延寿作用并不随培养基中虫草多糖浓度的提高而增强,只有在适当的浓度下,才能表现出显著的延寿效果.     

灵活操控果蝇翅芽中wingless基因表达水平的策略

【目的】灵活操控靶基因的表达水平对于研究基因的功能十分重要。Gal4/UAS系统已被广泛应用于调控基因表达,可研究果蝇Drosophila等模式生物复杂的生物学问题。受采用载体的特性及插入位点的影响,Gal4或UAS转基因品系在构建好之后,其调控靶基因的能力基本是确定的。本研究旨在在现有Gal4/UAS系统的基础上,开发一种新的策略,实现在果蝇翅芽中灵活操控wingless(wg)基因的表达水平。【方法】用遗传学手段将黑腹果蝇Drosophila melanogaster品系的UAS-wg和UAS-wg-RNAi转基因重组到同一黑腹果蝇品系中。将该重组黑腹果蝇品系与dpp-Gal4黑腹果蝇品系杂交,同时驱动UAS-wg和UAS-wg-RNAi在果蝇幼虫翅芽中共表达。杂交子代幼虫分别放置在不同的温度(18, 25和30℃)下培养。将幼虫翅芽解剖并进行免疫组化染色,测量染色的荧光强度,分析翅芽中wg的表达水平。【结果】在低温(18℃)下,UAS-wg在基因表达调控中起主要作用,wg表现为超表达,但其超表达的效率可被UAS-wg-RNAi有效地削弱。相反,在高温(30℃)下,UAS-wg-RNAi起主导作用,wg的表达受到抑制。并且通过转换温度,可实现wg在翅芽发育的不同阶段在超表达和抑制之间相互转化,从而灵活地操控wg基因在翅芽中的表达水平。【结论】该方法可以灵活操控果蝇翅芽中wg基因的表达水平,对于调控转基因的表达有重要的意义。     

人工蝉花孢梗束粗多糖的提取工艺和活性

通过单因素试验和正交试验研究了人工蝉花孢梗束粗多糖的提取工艺,并通过粗多糖对果蝇寿命、果蝇体内SOD活性及MDA含量的影响研究了粗多糖的活性。试验结果表明:人工蝉花孢梗束粗多糖的最佳提取工艺为液料比20∶1、时间2 h、温度90℃、浸提2次、醇沉乙醇浓度70%、醇沉时间为24 h;在该条件下的粗多糖得率为8.65%。孢梗束粗多糖能明显延长雌雄果蝇的寿命,延长率分别为18.78%和26.23%;孢梗束粗多糖能显著提高雌雄果蝇的SOD活性,并明显降低了雄性果蝇体内MDA含量,说明人工蝉花孢梗束粗多糖具有抗氧化活性和延长果蝇寿命的作用。     

异色瓢虫捕食胁迫对黑腹果蝇繁殖与后代发育的影响水

以模式昆虫黑腹果蝇(Drosophila melanogaster)及捕食性天敌异色瓢虫(Harmonia axyridis)为研究系统,通过把异色瓢虫接入到有黑腹果蝇的指形管中,研究了黑腹果蝇雌、雄成虫单独或共同被异色瓢虫捕食胁迫后,其自身寿命、子代的生长发育、繁殖和适合度的变化。结果表明:当雄性黑腹果蝇受到异色瓢虫成虫胁迫时,其寿命明显延长,但胁迫与否对雌性黑腹果蝇的寿命无明显影响;当黑腹果蝇的雌雄成虫同时受到胁迫时,其子代幼虫的发育历期延长,雄性后代寿命也增加,而雌性或雄性黑腹果蝇单独受到胁迫,其子代幼虫发育进度没有明显改变;当初孵黑腹果蝇幼虫受到异色瓢虫成虫直接或间接捕食胁迫后,其后续幼虫的发育历期都会受到影响,其中1龄期受到的间接捕食胁迫作用的影响大于直接捕食胁迫作用,直接捕食胁迫1龄幼虫比间接捕食胁迫更能延长其将来雌性成虫的寿命。     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

Attraction of Drosophila melanogaster males to food-related and fly odours

The fruit fly Drosophila melanogaster has become a model for olfaction and odour-mediated behaviour. In the wild, Drosophila flies aggregate on decaying fruit where they mate and oviposit and a strategy to find mates would be to locate fruit which has already been colonized by other flies. We therefore developed a bioassay to investigate attraction of males to food and fly odours. We showed that upwind flights are initiated by food odours. At shorter distances, males are attracted by volatiles produced by conspecifics. However, only odours produced by copulating flies attract males. This suggests either a synergistic effect of both male and female odours or changes in pheromone release during mating, that indicate the presence of sexually receptive females. Our findings demonstrate the essential role of food odours and pheromones for mate location in D. melanogaster.     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

Drosophila melanogaster larvae fed by glucose and fructose demonstrate difference in oxidative stress markers and antioxidant enzymes of adult flies

Activities of antioxidant and associated enzymes, and oxidative stress markers were assessed in newly enclosed adult fruit flies Drosophila melanogaster developed on diets with 4 and 10% glucose or fructose. In fly males, 10% fructose promoted higher content of protein carbonyls and catalase activity, but lower superoxide dismutase (SOD) activity than 4%, while in females-lower levels of high molecular mass thiols (H-SH). Females at all diets had virtually the same level of lipid peroxides, low-molecular-mass thiols, catalase, and superoxide dismutase activities. Fed with 4% fructose and glucose males demonstrated 24 and 26% lower H-SH level than females, respectively. On diets with 4% glucose, 10% glucose and fructose females had 32, 26 and 27% lower catalase activity than respective males, and 1.3-1.5-fold lower glucose-6-phosphate dehydrogenase activity on glucose-containing diets. Strong positive correlations between H-SH level and G6PD activity, as well as between catalase and G6PDH activity were found. These results suggest that type and concentration of dietary carbohydrate affect antioxidant defense in fruit flies. It also substantially depends on fly sex, comprising presumably levels of protein carbonyls and lipid peroxides, as well as catalase and SOD activities in males and G6PDH activity in females.     

Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila

The biguanide drug, metformin, commonly used to treat type-2 diabetes, has been shown to extend lifespan and reduce fecundity in C. elegans through a dietary restriction-like mechanism via the AMP-activated protein kinase (AMPK) and the AMPK-activating kinase, LKB1. We have investigated whether the longevity-promoting effects of metformin are evolutionarily conserved using the fruit fly, Drosophila melanogaster. We show here that while feeding metformin to adult Drosophila resulted in a robust activation of AMPK and reduced lipid stores, it did not increase lifespan in either male or female flies. In fact, we found that when administered at high concentrations, metformin is toxic to flies. Furthermore, no decreases in female fecundity were observed except at the most toxic dose. Analysis of intestinal physiology after metformin treatment suggests that these deleterious effects may result from disruptions to intestinal fluid homeostasis. Thus, metformin appears to have evolutionarily conserved effects on metabolism but not on fecundity or lifespan.     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系转化中的应用

[目的]通过检测黑腹果蝇 DDrosophiila melanogaster中piggyBac(PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力.[方法]构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体...     

短小乳杆菌的代谢产物对黑腹果蝇产卵选择性的影响

【目的】研究黑腹果蝇Drosophila melanogaster对短小乳杆菌Lactobacillus brevis有氧与无氧代谢产物的产卵选择,并解析这些代谢产物影响果蝇产卵行为的机制和生物学意义。【方法】应用产卵双选择装置,检测成年雌果蝇产卵的选择行为;利用黑暗条件(暗盒)、嗅觉突变体和摘除前足味觉感受器的方式探究介导该行为的感觉系统;通过在不同浓度乳酸(0, 70, 280 mmol/L)的培养基上培养黑腹果蝇,探究乳酸对其后代的发育历期和存活率的影响;利用免疫荧光染色法检测乳酸对果蝇肠道上皮细胞增殖情况的影响。【结果】短小乳杆菌无氧代谢产物引起成年雌性黑腹果蝇产卵避性,产卵指数为-0.47;短小乳杆菌有氧代谢产物乳酸含量为0.4 mmol/L,无氧代谢产物乳酸含量为126.8 mmol/L;雌性果蝇对乳酸同样产生产卵避性反应,且产卵避性随乳酸浓度的增高而增强。切除前足的果蝇对乳酸的产卵避性显著降低。与对照相比, 70 mmol/L的乳酸使幼虫的成蛹时间和成虫羽化时间分别延长了1.42 d和1.17 d,存活率降低了36.0%。乳酸明显破坏肠道上皮的完整性,促进肠道上皮细胞的增殖。【结论】短小乳杆菌无氧代谢产物引起成年果蝇产卵避性。果蝇主要通过味觉感知短小乳杆菌无氧代谢产物乳酸,这引起果蝇产卵避性,从而提高后代的生长发育和幼虫的存活率。     

基于mtDNA COI基因序列的中国柑橘果实蝇的分子鉴定及柑橘大实蝇种群的遗传多样性分析

【目的】探明危害我国柑橘的实蝇种类以及柑橘大实蝇Bactrocera minax不同地理种群和不同寄主种群的遗传多样性。【方法】利用mtDNA COI基因对危害柑橘的果实蝇进行种类鉴定,采用MEGA软件对其中28个地理种群的535头果实蝇COI基因片段(约505 bp)序列进行比对,分析种间及种内遗传距离,构建系统发育树。使用DnaSP软件分析柑橘大实蝇不同地理种群和不同寄主种群的遗传多样性。【结果】从柑橘虫果内共鉴定出4种实蝇,分别为柑橘大实蝇B.minax、桔小实蝇B.dorsalis、蜜柑大实蝇B.tsuneonis和瑞丽果实蝇B.ruiliensis。这4种实蝇的种间遗传距离为0.0264～0.2410,种内遗传距离为0.0000～0.0140,种间与种内遗传距离没有重叠区域。单个柑橘虫果内一般仅有1种实蝇,极个别柑橘果实可同时被两种实蝇危害(4/43);在这些为害柑橘的实蝇种类中,以柑橘大实蝇的个体数量比例最大,占90.70%。柑橘大实蝇地理种群遗传多样性高,28个种群共有17个单倍型。【结论】柑橘大实蝇是所调查地区柑橘实蝇的绝对优势种,其种群遗传分化程度较高,扩散危害风险大。本研究结果对柑橘果实蝇类的监测和防控具有重要意义。     

Biotin deficiency decreases life span and fertility but increases stress resistance in Drosophila melanogaster

Biotin deficiency is associated with fetal malformations and activation of cell survival pathways in mammals. In this study we determined whether biotin status affects life span, stress resistance, and fertility in the fruit fly Drosophila melanogaster. Male and female flies of the Canton-S strain had free access to diets containing 6.0 (control), 4.8, 2.5, or 0 pmol biotin/100 mg. Biotin concentrations in diets correlated with activities of biotin-dependent propionyl-CoA carboxylase and biotin concentrations in fly homogenates, but not with biotinylation of histones (DNA-binding proteins). Propionyl-CoA carboxylase activities and biotin concentrations were lower in males than in females fed diets low in biotin. The life span of biotin-deficient males and females was up to 30% shorter compared to biotin-sufficient controls. Exposure to oxidative stress reversed the effects of biotin status on survival in male flies: survival times increased by 40% in biotin-deficient males compared to biotin-sufficient controls. Biotin status did not affect survival of females exposed to oxidative stress. Exposure of flies to cold, heat, and oxidative stress was associated with mobilization of biotin from yet unknown sources. Biotin deficiency decreased fertility of flies. When biotin-deficient males and females were mated, the hatching rate (larvae hatched per egg) decreased by about 28% compared to biotin-sufficient controls. These findings are consistent with the hypothesis that biotin affects life span, stress resistance, and fertility in fruit flies.