DNA条形码技术在鉴定蛆症异蚤蝇中的应用

目的:建立应用DNA条形码技术鉴定蛆症异蚤蝇的方法。方法:分别提取在口岸检疫中截获的双翅目幼虫和成虫的基因组DNA,利用节肢动物线粒体细胞色素氧化酶I(COI)的通用引物进行DNA扩增。PCR产物直接测序,测序引物同扩增引物,得到清晰可读的665 bp COI的DNA片段碱基序列。与GenBank中相近的序列进行比较,并用ClustalW2在线建立NJ系统发育树。结果:成虫和幼虫COI的DNA序列100%相同,与GenBank中公布的长度为559 bp的蛆症异蚤蝇的COI相应片段相似性高达98.9%,与同属其他种类的可比片段的DNA序列相似性则低于90%。结论:根据结果可以判断所截获的双翅目昆虫为蛆症异蚤蝇。本文的研究再一次证明了DNA条形码技术可以直接应用于昆虫种类的鉴定。     

青岛胶东国际机场通航前捕获蚊蝇的分子鉴定

目的 了解青岛胶东国际机场转场前蚊类和蝇类的种群，为胶东机场病媒生物及病媒传播疾病的防控提供数据支撑。方法 2019年7-9月，在胶东机场口岸的草坪、人工湖、停机坪、宿舍区等4个区域，采用双层叠帐法、二氧化碳诱蚊灯法捕获成蚊，蝇笼法捕获蝇类，勺舀法捕获蚊类幼虫并进行自然孵育。利用形态学鉴定方法和基于线粒体COI基因的分子鉴定方法分类鉴定捕获的蚊类和蝇类。结果 2019年7-9月，捕获蚊类（含自然孵育）和蝇类分别为651只和2 599只。结合形态学和分子鉴定，蚊类1科4属6种，优势蚊种为中华按蚊、淡色库蚊和白纹伊蚊，占比分别为32.26%、27.65%和25.96%；蝇类4科12属13种，优势种为丝光绿蝇、大头金蝇和铜绿蝇，占比分别为31.67%、28.51%和21.51%。扩增蚊蝇的COI基因后，BLAST比对显示形态学与分子鉴定结果一致。结论 初步了解了青岛胶东国际机场转场前的蚊类和蝇类种群，为胶东机场通航后开展病媒生物监测提供了背景数据。     

应用DNA条形码技术鉴定未知双翅目蛹

目的建立应用DNA条形码技术鉴定未知病媒种类的方法,尤其是非成虫态虫体或者肢体残缺个体的DNA条形码鉴定的方法和技术,以缩短鉴定周期。方法提取从英国进口废纸中截获的双翅目单只蛹基因组DNA,利用多细胞无脊椎动物线粒体细胞色素氧化酶I(COI)的通用引物进行DNA扩增。PCR产物直接从两端测序,测序引物同扩增引物,序列经拼接后得到清晰可读的709bp COI基因片段碱基序列。同时将截获的同批蛹的其他个体在实验室内孵化成成虫进行形态学鉴定,并抽提孵化的成虫的基因组DNA,扩增其COI基因片段,测序并与蛹的序列进行比较。与GenBank中的序列进行同源性比对,并用Clustal W2在线建立AD系统发育树。结果未知种类的蛹和Genbank中红头丽蝇Calliphora vicina的COI DNA序列(包括引物序列)99.4%相同。蛹化成虫形态鉴定为红头丽蝇,孵化后的成虫的DNA条形码数据与蛹的数据完全相符。结论根据结果可以判断所截获的未知种类的蛹为红头丽蝇Calliphora vicina。本文的研究证明了DNA条形码技术是一种快捷准确的种类鉴定方法,尤其是运用在非成虫态的种类鉴定上。     

福建口岸三蚊种COI基因序列分析

〔目的〕分析福建口岸白纹伊蚊、中华按蚊和骚扰阿蚊细胞色素C氧化酶亚基I(COI)基因序列,为建立蚊种分子鉴定方法奠定基础。〔方法〕扩增并测定在福建口岸采集的白纹伊蚊、中华按蚊和骚扰阿蚊COI基因序列,与GenBank中相应序列进行对比分析。〔结果〕三蚊种COI基因扩增片段长度约500bp,A+T含量为65.61%~68.73%。核苷酸同源性分析结果表明,同一蚊种COI基因同源性为98.1%~100%,三蚊种间基因同源性为84.4%~87.7%;COI部分基因系统进化关系显示,三蚊种COI分子鉴定与形态学鉴定结果一致,阿蚊属与伊蚊属聚类为库蚊亚科,按蚊亚科位于系统进化树另一分支。〔结论〕COI基因核苷酸序列分析可应用于福建国境口岸三蚊种的区分,弥补形态特征信息量不足等传统分类鉴定方法的缺点,为建立外来或新发现蚊种分子鉴定方法奠定基础。     

辽宁口岸2001-2005年蝇类本底调查结果分析

目的掌握辽宁主要口岸蝇类本底状况,为蝇类防治提供技术支持。方法诱蝇笼法及网捕法。结果 2001-2005年,共捕获蝇类60968只,鉴定为5科43属101种。结论辽宁口岸地区,蝇科为蝇类优势种群,38种,占总种数的37.6%;其次为麻蝇科,28种,占总种数的27.7%。本次本底调查,初步了解辽宁口岸蝇类的种群构成,为辽宁口岸蝇类和相关虫媒传染病的防制、保障国境口岸的卫生安全提供技术支持。     

国内未见分布蝇种:澳洲麻蝇的形态和DNA条形码鉴定

目的 应用DNA条形码与形态分类相结合的方法,快速、准确鉴定在中山口岸入境船舶截获的1只蝇类.方法 2014年4月30日中山出入境检验检疫局神湾办事处从澳大利亚入境载有鲜葡萄的货船上截获1只蝇类,做成针插标本,并拍照存档.进行形态学鉴定,然后取1条后足提取基因组DNA,采用动物DNA条形码的通用引物(LCO1490和HCO2198)扩增目的片段,并进行纯化、克隆测序和序列分析,与NCBI及BOLD中的序列进行比对,构建NJ树.结果 形态学鉴定截获蝇类为雌性麻蝇科种类,由于缺少关于雌性麻蝇鉴定特征描述的参考资料,无法将该蝇鉴定到种,所获得的DNA条形码序列与BOLD中的澳洲麻蝇[Sarcophaga australis (Johnston et Tiegs,1921)]的序列相似度达100％.经过科技查新,确认是中国未见分布种.结论 根据鉴定结果,判定所截获的蝇类为雌性澳洲麻蝇,是我国首次截获.应用DNA条形码与形态鉴定相结合可以准确快速鉴定雌性麻蝇种类.     

基于COⅠ基因的媒介蝇类快速分子鉴定研究

目的 为解决蝇类形态学鉴定的不足,建立基于细胞色素C氧化酶亚基Ⅰ（COⅠ）基因的DNA条形码技术对蝇类进行分子鉴定。方法 选取烟台口岸常见的16只蝇类作为研究对象,形态学鉴定后,取蝇的单后足代表微量组织,提取总DNA。依据资料,选取COⅠ国际通用引物LCO1490：5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′,HCO2198：5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′,建立PCR体系,完善PCR程序,有效扩增蝇类的COⅠ基因。纯化PCR产物后进行序列测定和比对。此外,应用Mega5.05软件分别用最大似然法、邻接法、最大简约法分析系统发育,构建系统发育树比较进化关系。结果 16只蝇类COⅠ基因扩增片段长度为690 bp,其中A＋T平均含量高达68.53%。序列比对结果及系统进化关系显示,16只蝇类COⅠ基因的分子鉴定与形态学鉴定结果一致。结论 应用DNA条形码技术可成功扩增蝇类的COⅠ基因,其分子鉴定与形态学鉴定结果完全一致,尤其适用于微量组织样本的鉴定。     

2018年陕西口岸蝇类监测

目的掌握陕西咸阳机场口岸蝇类的种群分布,为口岸蝇类监测及防控提供科学依据。方法 2018年5-10月,每月在陕西咸阳机场口岸,采用笼诱法采集蝇类样本。结果监测共6天6次,捕获蝇类1 176只,经鉴定隶属于4科10属11种。优势种是棕尾别麻蝇、家蝇和丝光绿蝇。蝇类活动高峰出现在7～9月。结论掌握了陕西口岸蝇类构成,生活区和垃圾站是防治重点。     

2009年沈阳桃仙国际空港口岸蚊蝇种群及季节消长情况的调查

〔目的〕掌握沈阳桃仙国际空港口岸蚊、蝇种群、密度及季节消长情况,为媒介生物的防控及传染病监测提供科学依据。〔方法〕采用帐诱法捕蚊;笼诱法捕蝇。〔结果〕共捕获蚊类614只,经分类鉴定为2亚科3属7种;蝇类1188只,鉴定为7亚科10属15种;口岸蚊类以淡色库蚊和刺扰伊蚊为优势蚊种,分别占捕获总数的31.92%和29.15%;蝇类优势种为厩腐蝇,占捕获总数的33.50%,其次为黑尾黑麻蝇,占捕获总数的30.56%。〔结论〕本次调查较全面地掌握了沈阳口岸蚊蝇的本底及季节消长情况,为开展口岸医学媒介生物防控及传染病防治工作提供了科学依据。     

国境口岸常见11种蚊虫DNA条形码分子鉴定研究

目的通过研究国境口岸常见11种蚊虫的细胞色素C氧化酶亚基I(COI)基因序列差异性,并分析其系统进化关系,建立口岸常见蚊种的分子鉴定方法。方法设计l对扩增COI部分编码区的PCR引物,对广东、海南和云南等国境口岸采集的致倦库蚊、白纹伊蚊、中华按蚊等常见11种成蚊进行PCR扩增和序列测定,依据COI核苷酸序列进行系统进化分析。结果 11种蚊虫的COI基因扩增片断长度均为650 bp左右,A+T含量为66.51%~69.97%。同源性比较表明,同一蚊种间核苷酸序列同源性为94.8%~100.0%;不同蚊种间核苷酸序列同源性为77.7%~91.8%。系统进化关系显示,从种的水平上看,白纹伊蚊、致倦库蚊、中华按蚊等上述所有蚊种均聚集成簇,即同种之间呈明显的聚集,与形态学鉴定结果相一致。在属的水平上,库蚊属、阿蚊属、伊蚊属等均聚集成簇,形成各自的单独一支;各属间,阿蚊属与伊蚊属先聚类,再分别与库蚊属、曼蚊属聚类为库蚊亚科,按蚊属与上述蚊种亲缘关系较远。结论本次研究中的COI基因差异可作为国境口岸常见11种蚊虫分类鉴定的分子标记,为口岸常见蚊种的分子鉴定提供帮助,也可为口岸外来的或新发现的蚊种的鉴别提供分子水平的技术依据。     

中国不同地理株白纹伊蚊细胞色素C氧化酶亚基Ⅰ基因序列比较

目的：比较中国不同地理株白纹伊蚊细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列，从分子水平探讨不同地理株对登革病毒(DV)易感性的差异。方法：用聚合酶链反应从蚊虫基因组DNA扩增出COⅠ基因片段并进行克隆测序；用邻接法进行分子系统发育分析。结果：各地理株白纹伊蚊COⅠ基因片段序列长度均为415bp，所测各株序列均无缺失。云南思茅株的碱基转换率为1．93％，颠换率为0．24％。贵州麻尾株和广西南宁株的转换率为0．48％。各地理株中思茅株与麻尾株关系较近，麻尾株与南宁株关系较近，其余11个地理株均为同型株。结论：蚊虫对DV的易感性与多种因素有关，包括遗传及生态学方面(如季节气候、地理环境、人类活动等)的因素。在中国大多数白纹伊蚊为同型基因，其对DV的易感性尚无直接对应关系。     

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences.

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.     

Multiple lines of evidence on the genetic relatedness of the parthenogenetic and bisexual Haemaphysalis longicornis (Acari: Ixodidae)

As an obligate hematophagous ectoparasite, the hard tick Haemaphysalis longicornis exhibits two reproductive strategies, bisexual reproduction, and obligate parthenogenesis, which have attracted a widespread attention. However, the speciation of parthenogenetic population remained ambiguous due to its similarity in morphology but the remarkable differences in cytogenetics as compared with those of the bisexual ones. In the present study, we explored several new lines of genetic evidence to resolve this controversial issue. The number of the chromosomes in two lineages was checked by classical methods and their total DNA levels were determined utilizing flowcytometry. In addition, the sequences of 12S rDNA, 16S rDNA, cytochrome c oxidase I and II (COI, COII) and internal transcribed spacer-2 (ITS-2) genes were used to assess their phylogenetic relationship. We observed that the chromosome ploidy of bisexual and parthenogenetic H. longicornis collected by our laboratory was diploid and triploid, respectively. Flowcytometry analysis indicated a ratio close to 2:3 in the DNA contents of bisexual to parthenogenetic H. longicornis. Although the chromosome ploidy is different, their gene sequences are extremely similar. Analogous to the intra-species genetic difference of other invertebrates, sequence differences of all loci examined are below 2%. Phylogenetic trees constructed from 12S rDNA, 16S rDNA, COI, and ITS-2 genes revealed that they were all in the same monophyletic clade instead of splitting independently into evolutional branches. Moreover, according to 4× Rule, the K/θ ratio of two reproductive populations calculated based on COI was much smaller than four, strongly supporting that they belong to the same species. Therefore, we conclude that the evolutionary process just disturbs the chromosome ploidy and the sexual determination of parthenogenetic population and that it would be better to consider parthenogenetic H. longicornis as a metapopulation rather than a cryptic species.     

DNA条形码技术在未知昆虫幼虫种类鉴定中的应用

目的:建立用DNA条形码技术鉴定未知昆虫幼虫种类的方法,探讨DNA条形码在物种鉴定中的实用性。方法:利用多细胞无脊椎动物细胞色素氧化酶I(CO1)的通用引物进行PCR扩增,PCR产物克隆到pGM-T Vector中并测序,分别对同一种未知昆虫的成虫、幼虫的线粒体COI基因的709 bp片段进行序列分析,并与GenBank中的序列相比较。结果:测序结果未知种类的成虫和幼虫序列只有1个碱基的差异,一致性高达99.9%,与GenBank中的米象Sitophilus oryzae的序列一致性高达99.6%,与玉米象Sitophilus zeamais的序列一致性为86.6%。并与GenBank中其他种类的同一段序列相比较,用ClustalW-2构建系统关系树。以未知成虫的形态分类特征为验证,可以判断未知种类为米象。结论:结果表明运用DNA条形码技术能准确进行昆虫幼虫种类的鉴定。     

广东国境口岸不同蚊种COI序列分析和分子鉴定方法

[目的]建立国境口岸不同蚊种的细胞色素C氧化酶亚基I（COI）分子和氨基酸鉴定方法并分析其系统进化关系。[方法]设计1对扩增COI部分编码区的PCR引物，对广州机场、江门和湛江等国境口岸采集的致倦库蚊、三带喙库蚊、白纹伊蚊、中华按蚊、骚扰阿蚊等成蚊和实验室喂养的蚊幼虫进行PCR扩增和序列测定，分析COI核苷酸和氨基酸的系统进化关系。[结果]5种蚊种的COI基因扩增片断长度均为415bp，A＋T含量为68．77％-70．6％。同源性比较表明，不同蚊种间COI片断碱基变异颠换数都明显高于转化数，核苷酸序列及其编码的氨基酸序列同源性分别为85．1％-93．7％和92．0％-99.3％。COI核苷酸系统进化关系显示，所有蚊虫的COI分子鉴定与其形态学结果吻合，但骚扰阿蚊位于一单独的分支上，其亲缘关系与其它蚊种最远。COI基因编码的氨基酸系统进化与蚊虫形态学亲缘关系一致，库蚊属、伊蚊属、阿蚊属聚类为库蚊亚科，中华按蚊与其它蚊种的亲缘关系最远。[结论]建立的COI核苷酸和氨基酸鉴别技术可成功地应用于国境口岸范围内成蚊和幼蚊的属和种的区分，后者更能区分高级分类阶元亚科和正确反映蚊虫的系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点，为广东口岸和其它国境口岸范围内外来的或新发现的蚊种的鉴别提供了分子水平的技术依据。     

DNA条形码技术用于原木携带蚊种的鉴定

目的对临沂口岸进境原木上截获的蚊种进行DNA条形码研究确定蚊种。方法使用双翅目COI基因国际通用引物对原木携带蚊种COI基因进行PCR扩增和序列测定,序列在NCBI网站上进行Blast,确定蚊种。结果待测蚊种COI序列与GenBank中编号为HQ398903.1的骚扰阿蚊(Armigeres subalbatus)序列相似性为99%。结论基于COI基因的DNA条形码技术可成功地对媒介蚊类进行分类鉴定,该技术可弥补蚊虫形态缺失和形态学鉴定知识不足对蚊类鉴定的影响。     

DNA条形码技术在贵州茂兰鼠类鉴定中的应用

目的探讨基于线粒体COI基因的DNA条形码技术对鼠类进行准确的鉴定。方法以贵州茂兰地区的3个常见鼠种21个鼠类标本为研究对象,提取基因组DNA,扩增线粒体细胞色素C氧化酶亚基Ⅰ(COI)基因片段并测序,将测序结果进行BLAST比对,基于双参数模型计算鼠种种间和种内遗传距离,构建分子发育系统。结果 DNA条形码检测技术可用于该地区鼠种的鉴定,并能纠正现场形态学鉴定的失误。结论研究结果表明DNA条形码技术是一种有效可靠、易于操作的鼠类鉴定方法,能纠正形态学鉴定中的错误。     

DNA条形码技术应用于口岸吸血蠓类快速分子鉴定的研究

目的建立口岸吸血蠓类快速分子鉴定方法,提高鉴定效率和准确率。方法选择口岸区域采集点,采集吸血蠓类。经形态学鉴定后,单只蠓提取基因组DNA,采用自行设计的COI基因扩增引物进行扩增。PCR产物经电泳鉴定后,进行双向测序。序列经分析后,去除引物序列和不稳定序列,进行拼接。提交至NCBI和BOLD数据库进行比对,并建立系统进化树进行验证。结果得到12个新序列,已登录至GenBank,登录号为KF528689~KF528700。其中台湾蠛蠓的COI序列(683 bp)填补了NCBI数据库的空白。所得序列经分析后可以将多种蠛蠓、库蠓很好地区分开。结论本项目所建立的基于吸血蠓类COI基因的DNA条形码快速分子鉴定方法可以很好地鉴定各吸血蠓种,值得进一步推广应用。     

Analysis of minicircle classes in Leishmania (Viannia) species

This paper reports the analysis of minicircle sequence classes from 4 Leishmania species, all belonging to the 'New World' species of the subgenus Viannia: Leishmania braziliensis, L. guyanensis, L. panamensis and L. peruviana. A minicircle library was constructed for each species, and clones were analysed by restriction enzyme digest and sequence analysis. 319 minicircles from the 4 species were examined and 96 of these were wholly or partially sequenced. The sequences of 41 whole minicircles--21 from L. panamensis, 8 from L. guyanensis and 6 each from L. braziliensis and L. peruviana are presented. Sequence classes were identified within which sequences were highly conserved, with only a small number of single base pair changes between them. In contrast, minicircles from different classes differed significantly, displaying sequence homology only over the minicircle conserved region. Some minicircle classes were identified which were shared between species. These minicircles displayed sequence variation which was potentially species-specific, and were analysed phylogenetically. These results question the hypothesis that minicircle sequence is rapidly evolving and also suggest that an as yet unknown selective pressure maintains sequence class conservation over the entire minicircle molecule even in different species, not only over the conserved region and the guide ribonucleic acid gene. A novel hypothesis is proposed to explain these results.     

A new species of tick,Ixodes (Ixodes) mojavensis (Acari: Ixodidae), from the Amargosa Valley of California

Ixodes (Ixodes) mojavensis, n. sp. (Acari: Ixodidae), is described from all parasitic stages collected from the endangered vole Microtus californicus scirpensis Bailey, 1900 (Rodentia: Cricetidae), Mus musculus L. 1758 (Rodentia: Muridae), and Reithrodontomys megalotis (Baird; 1857) (Rodentia: Cricetidae) in the Amargosa Valley of California. When first collected in 2014, this tick was tentatively identified as Ixodes minor Neumann, 1902 because the nucleotide similarity between its 16S rDNA sequence and a homologous GenBank sequence from an I. minor from the eastern U.S. was 99.51%. Nevertheless, adults of I. mojavensis differ morphologically from I. minor by hypostomal dentition, absence of a spur on palpal segment I, and punctation patterns; nymphs by the shapes of basis capituli, auriculae, cervical grooves and external files of hypostomal denticles; and larvae by the length of idiosomal setae and hypostomal dentition. DNA sequencing of fragments of 4 different genes, 12S rDNA, 16S rDNA, cytochrome c oxidase subunit I (COI), and intergenic transcribed spacer 2 (ITS2) of I. mojavensis and of closely related species of Ixodes shows that the mitochondrial gene sequences of the new tick species are almost identical to the I. minor homologous genes. Phylogenetically, the two species do not cluster in mutually exclusive monophyletic clades. However, ITS2 sequences of I. mojavensis and I. minor diverge deeply (≥ 5.74% maximum likelihood divergence) and are as different as homologous genes from other recognized species. The discrepancy between the two sets of genes is suggestive of past mitochondrial introgression or incomplete mitochondrial lineage sorting.