三种革兰阴性细菌在按蚊饲养水体及按蚊中肠内存活能力的研究

目的 探讨分离自中华按蚊雌成蚊中肠的3种革兰阴性细菌,即气单孢菌、A4菌(未鉴定)和肠杆菌在按蚊幼虫孳牛水体中及按蚊体内的存活能力. 方法 3种目标细菌在实验室经数量扩增后,添加到饲养按蚊幼虫的水体中,依次收集饲养盆和蛹盒内的水样品,并解剖羽化后吸血雌成蚊,取其中肠制成研磨液;建立水样品和研磨液中细菌的16S rDNA序列克隆文库,并从各样品中分离可培养细菌,比较各样品中的优势菌种和不同处理中可培养细菌的种类组成. 结果 在所有样品中均未检测到与加入的人工培养细菌种类相同的细菌.按蚊幼虫饲养水体和蛹盒水体中的可培养细菌均以嗜水气单孢菌和睾丸酮丛毛单胞菌为主,在少量按蚊的中肠内含有可培养细菌,但均不是目标细菌;而各处理样品中的优势菌均为铜绿假单孢菌. 结论 3种细菌经人工培养后,不能在按蚊幼虫生活水体中建立种群,导致未能在按蚊成蚊中肠内柃测到这些细菌.     

三种革兰阴性细菌在按蚊饲养水体及按蚊中肠内存活能力的研究

目的 探讨分离自中华按蚊雌成蚊中肠的3种革兰阴性细菌,即气单孢菌、A4菌(未鉴定)和肠杆菌在按蚊幼虫孳牛水体中及按蚊体内的存活能力. 方法 3种目标细菌在实验室经数量扩增后,添加到饲养按蚊幼虫的水体中,依次收集饲养盆和蛹盒内的水样品,并解剖羽化后吸血雌成蚊,取其中肠制成研磨液;建立水样品和研磨液中细菌的16S rDNA序列克隆文库,并从各样品中分离可培养细菌,比较各样品中的优势菌种和不同处理中可培养细菌的种类组成. 结果 在所有样品中均未检测到与加入的人工培养细菌种类相同的细菌.按蚊幼虫饲养水体和蛹盒水体中的可培养细菌均以嗜水气单孢菌和睾丸酮丛毛单胞菌为主,在少量按蚊的中肠内含有可培养细菌,但均不是目标细菌;而各处理样品中的优势菌均为铜绿假单孢菌. 结论 3种细菌经人工培养后,不能在按蚊幼虫生活水体中建立种群,导致未能在按蚊成蚊中肠内柃测到这些细菌.     

中华按蚊实验室种群中肠内细菌菌群分析

目的为获取实验室饲养的3个发育期中华按蚊中肠内的革兰氏阴性菌菌株。方法取实验室饲养的20只中华按蚊吸血成蚊的中肠,研磨稀释后涂板培养,挑取单个菌落,培养扩增。以各菌株的DNA为模板,对16SrRNA基因的V3高变区进行PCR扩增、测序和Blast分析。对经鉴定的各类细菌进行革兰氏染色。以中华按蚊的Ⅳ龄幼虫(10只)、刚羽化雌成蚊(50只)和吸血雌成蚊(20只)等3个发育期的中肠基因组DNA为模板,用变性梯度凝胶电泳技术(DGGE)分析3个发育期中肠细菌16SrDNAV3区序列;建立在3个发育期均存在的16SrDNAV3区的克隆文库,并进行测序分析。将两种方法获得的16SrDNAV3区序列进行亲缘关系分析,确定同种的革兰氏阴性菌。结果从吸血成蚊的中肠分离到28个菌株,经分类鉴定为5种细菌,除短芽孢杆菌革兰氏染色呈阳性外,气单孢菌、丛毛单胞菌、金黄杆菌和A4菌均为革兰氏阴性菌。DGGE分析显示,共检测到4组条带在3个发育期均存在,经序列分析来源于5种细菌,分别为气单孢菌、A4菌、假单胞菌、无色杆菌和某未知菌种,其中气单孢菌(GQ301543)和A4菌(FJ8701127)来源的16SrDNAV3区与分离得到的该两种菌株的序列一致性均为100%。结论获得2种在中华按蚊3个发育期中肠内均存在的革兰氏阴性菌株,分别为气单孢菌和A4菌。     

按蚊中肠细菌及其应用的研究进展

按蚊中肠细菌能够在控制传疟按蚊种群数量和抑制疟原虫在按蚊体内的发育等方面发挥重要作用。本文从细菌与按蚊的食物关系、按蚊幼虫和成蚊中肠细菌及其应用、细菌在按蚊体内的经发育期传递、细菌与按蚊成蚊的免疫反应,以及利用细菌防控疟疾流行的可行性等方面进行综述。     

白纹伊蚊不同发育阶段可培养共生细菌多样性研究北大核心CSCD

目的探讨白纹伊蚊不同发育期可培养共生细菌的多样性。方法培养白纹伊蚊不同发育期共生菌,根据菌落形态特征和16S rDNA序列的系统发育分析,判定分离的可培养细菌种类。结果共分离到17株细菌,隶属于4个门、8个科、14个属,其中变形菌门是优势菌群;幼虫、蛹和雌蚊中分别分离到7、8、8株细菌,且3个发育期各分离到4个特有菌株。结论白纹伊蚊体内可培养共生细菌具有多样性,可为利用细菌进行蚊虫生物防制提供基础资料。     

经Bisazia绝育的大劣按蚊感染恶性疟原虫的实验研究

以实验室内饲养的大劣按蚊产生的蛹作试验。蚊蛹用绝育剂Bisazia(ENf-6158)即p,p,-bis(1-氮丙啶)-N-甲基膦硫代酰胺处理。将Bisazia配成0.125、0.25、1.0、1.5和2.0%不同浓度的水溶液,在盛有100ml上述溶液的容器中,各投入蚊蛹250只,60分钟后取出用清水漂洗10分钟,而后移入清水,置蚊笼内待其羽化为成蚊。经绝育剂处理后羽化的成蚊,分绝育雌蚊×绝育雄蚊,正常雌蚊×绝育雄蚊,绝育雌蚊×正常雄蚊,以及正常雌蚊×正常雄蚊等     

嗜人按蚊生物学的实验室研究

选用引自江苏的嗜人按蚊实险室传代品系,在26±1℃,相对湿度70±10%,每日光照12h条件下,卵、幼虫及蛹期分别为2.93±0.60d,7.98±0.87d和1.70±0.34d。孵化、化蛹及羽化率分别为82.70%、72.20%和96.88%。成蚊雌雄比例约为1:1。喂血雌蚊、喂糖水雌蚊和雄蚊的寿命分别为28.45±8.92d、17.27±5.45d和11.93±3.56d。雌蚊平均受精率为79.98d±10.40%;羽化后d3～6累计吸血率为86.67%。群体观察每雌蚊一生平均产卵265.17粒,较单个饲养产卵量为多。产卵高峰时间集中在21:00～23:00,此时产卵量约占昼夜产卵总数的78.33%。     

巴拉巴按蚊复合体未成熟期形态测量的多元分析

本文报道用判别式函数的多元分析方法,检查巴拉巴按蚊复合体中三株幼虫与蛹的形态,并订出鉴定的客观标准。三株蚊虫均保存于伦敦卫生学与热带医学院的Ross研究所内,根据其染色体的类型而分为:(1) 大劣按蚊X_A株(来自泰国的巴页河省);(2) 大劣按蚊X_B株(来自马来西亚的玻璃市);(3) 巴拉巴按蚊X_C株(来自沙巴)。取上述三株已交配雌蚊所产的卵,培育后制成幼虫皮与蛹皮标本,并检查其四龄     

微小按蚊和溪流按蚊翅斑形态的比较

目的：比较微小按蚊和溪流按蚊翅斑的形态，了解它们的变异情况，寻找可靠的鉴别特征。方法：微小按蚊采自云南省景洪县基诺乡，溪流按蚊采自广西壮族自治区凌云县。将母蚊产的卵，在实验室饲养至成蚊，再将成蚊的翅制成玻片标本，在显微镜下测量标本的翅长和部分翅斑的长度。结果：共检查微小按蚊雌蚊翅５２个，雄蚊翅６０个，溪流按蚊雌蚊翅４０个，雄蚊翅６０个。认为翅前缘脉上分脉前白斑（ＰＳＰ）的有无和分脉白斑与分脉暗斑比例（ＳＰ／ＳＤｒａｔｉｏ）的大小，是鉴别微小按蚊和溪流按蚊的重要依据。结论：无论是雌蚊还是雄蚊，微小按蚊的翅斑形态与溪流按蚊的翅斑形态均有较为明显的差异。     

改进某些按蚊多线染色体的制备技术

本文报道按蚊多线染色体制备的一些改进。作者认为已有的技术(French 等,1962,Kanda,1971)不能适用于按蚊、果蝇和摇蚊的所有种类,因此对染色体的制备和观察技术作了若干改进。要得到良好的多线染色体制备,必须要有好的幼虫材料。这些材料可以直接从野外采集健康的四龄幼虫,也可以诱捕野外的孕卵雌蚊,待它产卵孵化后,饲养到四龄时应用。幼虫用混合饲料饲养。喂1～2龄幼虫     

中华按蚊细胞色素P450基因表达特征分析

目的 探讨6种溴氰菊酯抗性候选细胞色素P450（CYP）基因（CYP6M3、CYP6Y1、CYP6P5、CYP4H14、CYP4G17、CYP12F16）在中华按蚊体内的表达特征。方法 收集中华按蚊不同发育时期（卵、幼虫、蛹、雌性成蚊和雄性成蚊）和组织（唾液腺、马氏管、中肠、卵巢以及脂肪体）样本,以及雌性成蚊在暴露于不同溴氰菊酯剂量（0、1.25、3.75、6.25、12.5 μg/瓶）和时间（0,5、15、30、60 min）后的样本.提取总RNA,利用反转录实时定量PCR（qPCR）技术分析CYP6M3、CYP6Y1、CYP6P5、CYP4H14、CYP4G17、CYP12F16基因在中华按蚊不同发育时期、组织以及不同溴氰菊酯接触剂量和时间下的相对表达量。结果 CYP6M3与CYP6Y1基因在雄性中华按蚊成蚊体内的表达量最高,CYP6M3基因在雄性成蚊体内的表达量是雌性成蚊的35.1倍,CYP6Y1基因在雄性成蚊体内的表达量是雌性成蚊的61.4倍;CYP4H14基因在幼虫期表达量最低,且在雌性成蚊体内表达量是四龄幼虫体内表达量的22.5倍。候选CYP基因在中华按蚊不同组织内的表达量差异具有统计学意义,CYP6M3基因在马氏管内的表达量是在卵巢中的38.9倍,CYP6Y1基因在脂肪体内的表达量是在卵巢中的9.1倍,CYP6P5基因在中肠内的表达量是在卵巢中的30.3倍,CYP4G17基因在脂肪体内的表达量是在卵巢中的4.6倍,CYP12F16基因在马氏管内的表达量是在卵巢中的4.4倍。接触不同溴氰菊酯剂量和时间对候选CYP基因的表达水平表现出一定的诱导效应,影响候选CYP基因在中华按蚊体内的表达。结论 候选CYP基因在不同发育期中华按蚊体内和不同组织中差异表达,暴露于不同溴氰菊酯剂量和时间影响CYP基因在中华按蚊体内表达。     

Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water

In a paratransgenic approach, genetically modified bacteria are utilized to kill the parasite in the vector gut. A critical component for paratransgenics against malaria is how transgenic bacteria can be introduced and then kept in a mosquito population. Here, we investigated transstadial and horizontal transfer of bacteria within an Anopheles gambiae mosquito colony with the focus on spiked breeding sites as a possible means of introducing bacteria to mosquitoes. A Pantoea stewartii strain, previously isolated from An. gambiae, marked with a green fluorescent protein (GFP), was introduced to mosquitoes in different life stages. The following life stages or older mosquitoes in the case of adults were screened for bacteria in their guts. In addition to P. stewartii other bacteria were isolated from the guts: these were identified by 16S rRNA sequence analysis and temporal temperature gradient gel electrophoresis (TTGE). Bacteria were transferred from larvae to pupae but not from pupae to adults. The mosquitoes were able to take up bacteria from the water they emerged from and transfer the same bacteria to the water they laid eggs in. Elizabethkingia meningoseptica was more often isolated from adult mosquitoes than P. stewartii. A bioassay was used to examine An. gambiae oviposition responses towards bacteria-containing solutions. The volatiles emitted from the solutions were sampled by headspace-solid phase microextraction (SPME) and identified by gas chromatography and mass spectrometry (GC-MS) analysis. P. stewartii but not E. meningoseptica mediated a positive oviposition response. The volatiles emitted by P. stewartii include indole and 3-methyl-1-butanol, which previously have been shown to affect An. gambiae mosquito behaviour. E. meningoseptica emitted indole but not 3-methyl-1-butanol, when suspended in saline. Taken together, this indicates that it may be possible to create attractive breeding sites for distribution of genetically modified bacteria in the field in a paratransgenic approach against malaria. Further research is needed to determine if the bacteria are also transferred in the same way in nature.     

Polymerase chain reaction-based identification and genotyping of Anopheles mosquitoes with a 96-pin bacterial replicator

A simple method for rapid identification of large numbers of Anopheles mosquitoes was developed based on polymerase chain reaction (PCR) amplification of the rDNA intergenic spacer and internal transcribed spacer 2. By means of previously described primers for the Anopheles gambiae and An. quadrimaculatus species complexes, rDNA was amplified simultaneously from 96 whole mosquitoes or parts. No homogenization or individual DNA preparation was necessary, and transfer of 96 samples to PCR reactions was performed simultaneously with a bacterial replicator. Control reactions indicate that the level of cross-contamination is negligible, and false-negative findings are rare. The method was tested on larvae, pupae, adult heads, whole adult males and females, and single tarsi. All parts except tarsi provided satisfactory template. Fresh, ethanol-preserved, dried, and frozen adults were also tested with similar results. The method was also tested for amplification of a single-copy gene, white. Results were generally positive, although some false-negative findings were observed. This method allows rapid analysis of large numbers of mosquitoes without robotic equipment and should enable rapid and extensive PCR analysis of field-collected samples and laboratory specimens.     

16s rDNA序列分析在腹泻粪便标本菌群分析应用

目的应用16s rDNA序列分析方法,对临床实验室没有分离到已知病原菌的腹泻病人粪便标本进行菌群分析,为腹泻病人的病原学诊断提供线索。方法根据细菌16s rDNA保守区序列设计通用引物,以腹泻病人粪便标本中的总DNA为模板,PCR扩增16s rDNA片段。将获得的片段克隆入T载体进行测序,与数据库中发表序列的进行比对,判断标本中细菌的种类和比例;PCR扩增常见的五类致病性大肠杆菌的特征基因应用于排除诊断。结果在3份粪便标本中,大肠杆菌为优势菌群(所占比例分别为84%、65%和81%);其他种群细菌为拟杆菌属(Bacteroides)、柠檬酸杆菌属(Citrobacter)和普雷沃氏菌属(Prevotella)等;实验室常规培养方法没有分离到常见的五类致病性大肠杆菌;PCR扩增没有发现常见的五类致病性大肠杆菌的特征基因。结论引起三例病人腹泻的病原菌可能是一种新的大肠杆菌;16s rDNA序列分析方法可应用于腹泻粪便标本菌群分析,为常规的病原培养方法提供补充,对疾病的诊断提供线索。     

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97--100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.     

Intraspecific Genetic Variation and Phylogenetic Analysis of Dirofilaria immitis Samples from Western China Using Complete ND1 and 16S rDNA Gene Sequences

Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1.     

Bacterial biota in the human distal esophagus

The esophagus, like other luminal organs of the digestive system, provides a potential environment for bacterial colonization, but little is known about the presence of a bacterial biota or its nature. By using broad-range 16S rDNA PCR, biopsies were examined from the normal esophagus of four human adults. The 900 PCR products cloned represented 833 unique sequences belonging to 41 genera, or 95 species-level operational taxonomic units (SLOTU); 59 SLOTU were homologous with culture-defined bacterial species, 34 with 16S rDNA clones, and two were not homologous with any known bacterial 16S rDNA. Members of six phyla, Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Fusobacteria, and TM7, were represented. A large majority of clones belong to 13 of the 41 genera (783/900, 87%), or 14 SLOTU (574/900, 64%) that were shared by all four persons. Streptococcus (39%), Prevotella (17%), and Veilonella (14%) were most prevalent. The present study identified approximately 56-79% of SLOTU in this bacterial ecosystem. Most SLOTU of esophageal biota are similar or identical to residents of the upstream oral biota, but the major distinction is that a large majority (82%) of the esophageal bacteria are known and cultivable. These findings provide evidence for a complex but conserved bacterial population in the normal distal esophagus.