Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method.

Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis, I. riparia and I. anglicana, irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases TaqI, VspI, MvaI and Bsp143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, VspI and Bsp143I appear to be an appropriate combination.     

亚洲部分地区东方蜜蜂mtDNA多态性研究

 就云南省7个不同样点共7群东方蜜蜂蜂群开展mtDNA内切酶位点测试和分析研究,并与菲律宾的东方蜜蜂作对比分析。内切酶DraI分析7个蜂群,共发现4个内切酶位点;云南东方蜜蜂mtDNA的tRNAleu-COII基因的序列没有长度多态性,EcoRI内切酶位点共有4个,与菲律宾的吕宋岛的东方蜜蜂属于同一单倍型。另外,对来自尼泊尔、泰国、越南、老挝以及我国北京、云南等地的共13群蜜蜂mtDNA COII基因的420个碱基对进行测序分析,发现13群东方蜜蜂分析的420个碱基序列里,位点变异从0个到5个不等,变异率从0到1.19％. 用邻接法(neighbor-joining)构建的分子系统树可以看出分枝树主要分为两个类群:高纬度或高海拔的蜂群为一个类群,它们主要来自撒营盘（2）（China 4）、丽江(China 7)、北京(1)（China 1）、尼泊尔（Nepal）、北京（2）（China 2）、河口（1）（China 8）和越南（2）（Vietnam 2）。另一个类群的主要来自低纬度或低海拔的蜂群,它们主要来自撒营盘（1）（China 3）、景洪（China 5）、越南（1）（Vietnam 1）、泰国（Thailand）、河口(2) (China 6)和老挝(Laos)。该实验为我国东方蜜蜂资源的保护和开发研究提供了重要的信息和手段。     

High levels of genetic differentiation between Wolbachia-infected and non-infected populations of Folsomia candida (Collembola, Isotomidae)

The Gram-negative -Proteobacterium Wolbachia pipientis has been described as an obligate endosymbiont in many arthropod species, where it induces a variety of reproductive alterations, including parthenogenesis. Recently, this microorganism has also been detected in the parthenogenetic collembolan Folsomia candida. Here, we confirm the occurrence of the endosymbiont also in two Italian parthenogenetic populations of F. candida using ultrastructural (electron microscopy) and molecular (PCR screening on two bacterial genes) evidence. The strain isolated in the Italian populations has almost-identical gene sequences compared to that previously isolated in other populations of F. candida. In addition, we discovered a population of Folsomia cf. candida, which showed the presence of both males and females. This population is not infected by Wolbachia. A screening of two mitochondrial genes (COI and COII) showed that the bisexual population has high levels of genetic divergence in comparison with the parthenogenetic ones, even suggesting the possibility that it belongs to a different species. Furthermore, the remarkably high levels of genetic divergence between the two parthenogenetic populations suggests a possible influence of Wolbachia on inducing such differentiation, and, in the long term, speciation.     

Identification of two haplotypes of cytochrome oxidase subunit II (COII) gene of the Formosan subterranean termite,Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), in southern Mississippi

BACKGROUND: Coptotermes formosanus Shiraki was accidentally introduced into the United States from Asia. The introduction of the pest has brought significant economic consequences. During the past decade, Mississippi has become a significantly infested state, partly due to the proximity to coastal port cities such as New Orleans. This study was initiated to investigate the origin and infestation route of C. formosanus in southern Mississippi. RESULTS: Twenty‐eight colonies (21 colonies from Mississippi, six from Louisiana, one from China) were collected. Sequencing and analysis of 112 sequences revealed 15 haplotypes of cytochrome oxidase subunit II (COII) gene in the world. Two haplotypes of COII were identified in Mississippi. In addition, specific primers were designed and tested differentially to amplify characteristic fragments for verifying and surveying different genotypes of C. formosanus in the future. CONCLUSION: Of the two haplotypes identified in Mississippi, the GA group was identical to those reported previously in Georgia, Louisiana, Alabama and other infested states. The second haplotype, the AT group, was identified for the first time in southeastern United States. Sequence identity of the AT‐group C. formosanus with those reported mainly in southeastern Asian countries provided evidence of at least two introductions of C. formosanus into the United States. Copyright © 2010 Society of Chemical Industry     

基于线粒体COII和ND4基因序列的塔里木裂腹鱼群体遗传多样性分析

为探究塔里木裂腹鱼群体的遗传多样性和遗传分化现状,选用车尔臣河、克孜勒河和阿克苏河3个不同地理种群的塔里木裂腹鱼共计126尾样本,进行线粒体DNA COII和ND4基因序列的对比分析,探讨了2种标记对塔里木裂腹鱼遗传多样性分析结果的差异和关系。结果显示,塔里木裂腹鱼线粒体DNA COII和ND4基因序列的A+T含量均高于G+C含量,碱基组成具有偏倚性。基于线粒体DNA COII和ND4基因序列分析,126个样本中分别确定了6个和23个单倍型,其中线粒体DNA COII基因序列中3个群体存在共享单倍型现象,线粒体DNA ND4基因序列中未发现3个群体间存在共享单倍型。2种标记下群体的单倍型多样性和核苷酸多样性分别为0.738±0.019/0.904±0.014和0.02129±0.00298/0.04876±0.00149,呈现出高单倍型多态性和高核苷酸多态性的特征。2种标记的分子方差分析（AMOVA）均表明,在所有群体中遗传变异主要来源于群体间,且车尔臣河群体的塔里木裂腹鱼与其他2个群体的遗传分化均达到显著水平（P<0.05）。贝叶斯法（BI）构建的BI树与单倍型网络图结构一致,塔里木裂腹鱼形成了2个分支。COII和ND4基因序列的岐点分布图均呈现双峰型,表明塔里木裂腹鱼现在的分布是先前分化种群发生二次交流的结果。种群结构分析结果亦表明塔里木裂腹鱼已分化出2个明显的地理种群,故建议将车尔臣河群体作为塔里木裂腹鱼的亚种。     

M、F型COII基因在雌雄三角帆蚌不同发育时期的表达差异研究

与传统的母系遗传不同,双壳贝类线粒体有M和F型2种mt DNA,称为双单亲遗传(doubly uniparental inheritance,DUI)。为了探究DUI的形成机制,实验利用RACE方法得到三角帆蚌M、F-type COⅡ基因的cDNA全长;荧光定量检测各时期各组织中M、F-type COⅡ基因的表达。结果发现,(1)M-type COⅡ基因的cDNA序列全长1244 bp,F-type COⅡ基因的cDNA序列全长808 bp,M型3′端较F型有一段546 bp的特异性延长序列;跨膜结构预测显示M-type COⅡ基因3′特异性延伸区域有4个跨膜结构,推测这段延伸序列具有一定的功能性;(2)早期幼龄(胚胎期-5月龄)三角帆蚌的组织团中,M、F-type COⅡ基因均能检测出,且同时期中F-type COⅡ基因的表达量显著高于M-type COⅡ;(3)6月龄蚌各组织中,F-type COⅡ基因在各组织中表达量明显高于M型的表达量,而M-type COⅡ基因在性腺中表达明显高于其他组织;(4)一龄雌性各组织中,M、F-type COⅡ基因均有表达;而一龄雄性各组织中,M-type COⅡ基因仅在性腺中表达;(5)二龄雌性各组织中,M、F-type COⅡ基因均有表达;二龄雄性各组织中,F-type COⅡ基因除性腺外的其他组织中均有低量表达,M-type COⅡ基因仅在性腺中表达,且表达量较高。研究表明,在三角帆蚌雌雄个体发育过程中,M-type mt DNA在组织中选择性降解或聚集。     

Molecular phylogeny of three subfamilies of the Neanuridae (Insecta, Collembola) and the position of the antarctic species Friesea grisea Schäffer.

The nuclear 28S rRNA and the mitochondrial COII gene were used to establish phylogenetic relationships among species of the family Neanuridae, with special emphasis on species of the subfamily Neanurinae. Phylogenetic analysis was conducted using genetic distances, parsimony and likelihood methods. The D3-D5 fragment of the rRNA gene was very conserved, both in sequence and in secondary structure features. This fragment supplied little information on relationships at this level. The phylogenetic reconstruction based on 1st and 2nd codon positions of the COII gene was partly in accordance with morphological data, but it was discordant for the placement of some species. Relationships among the subfamilies Frieseinae, represented by the Antarctic species Friesea grisea, Pseudachorutinae and Neanurinae were uncertain. The subfamily Neanurinae and its tribes Neanurini and Paleonurini were shown as monophyletic taxa. Relationships between three species of the genus Bilobella were in accordance with morphological and biochemical data. Relationships between genera within the Neanurini were more controversial. In accordance with morphological hypotheses, a basal position of Thaumanura was suggested, but the molecular data placed Neanura muscorum in a derived position, in sharp contrast with morphological evidence. A close relationship was suggested between Deutonura conjuncta, Cansilianura malatestai and Lathriopyga longiseta. The disagreement between molecular and morphological data suggests that one or both data sets might be affected by a certain degree of homoplasy and that these data should be interpreted with caution in phylogenetic reconstructions.     

细鳞斜颌鲴三个群体线粒体COⅡ基因的遗传变异

应用线粒体COⅡ基因序列分析了细鳞斜颌鲴(Plagiognathops microlepis)三个群体(梁子湖群体、龙窝湖群体和淮河群体)的遗传变异关系.结果显示:测序得到细鳞斜颌鲷线粒体COⅡ基因大小为681 bp,A+T含量(56％左右)明显高于G+C含量.三个群体18个个体共测出5种单倍型,42个核苷酸变异位点,...