DNA条形码技术在福建省蝇类鉴定中的应用

目的探讨DNA条形码技术应用于福建省蝇类鉴定的可行性,并初步建立福建省蝇类的基因数据库。方法采集自福建省部分地区的4科13属19种64个蝇类新鲜标本,提取基因组DNA进行PCR扩增,序列测序,并进行同源性对比、进化距离分析,构建系统进化树。结果基因序列分析结果显示,上述蝇类mt DNA上细胞色素C氧化酶亚基Ⅰ基因序列的种内进化分歧均数在0～3.32%之间;除铜绿蝇与丝光绿蝇进化分歧均数为0.69%外,其余种间进化分歧均数在5.26%～18.30%之间;系统进化树分析显示,相同种能够很好聚集在一起,Bootstrap检验可信度均达96%以上。结论DNA条形码技术可用于福建省一些常见蝇类的鉴定,作为形态学鉴定的补充手段。     

DNA条形码识别——DNA条形码与DNA芯片识别蚊媒准确性的比较

〔目的〕比较DNA条形码和DNA芯片这2种技术平台在口岸医学媒介蚊类识别中的准确性。〔方法〕以20种媒介蚊类的线粒体COI基因序列信息为分析对象,采用NJ邻接法计算公认的DNA条形码区域在物种识别中的准确性,采用DNA芯片技术分析2种长度探针在物种水平识别中的差异。〔结果〕DNA条形码的准确性为98.5%,数据集中95%物种的唯一序列最短长度为50bp左右;DNA芯片的准确性为90%～98.4%,25bp探针比55bp探针更具优势。〔结论〕DNA条形码和DNA芯片都能够准确地识别20种媒介蚊类,DNA条形码芯片技术有望在口岸出入境检验检疫领域得到应用。     

新截获中国未见分布种杜比亚丽蝇形态和DNA条形码鉴定

目的利用DNA条形码技术鉴定外来媒介生物,提高物种鉴定的速度与准确度。方法对2013年9月在中山神湾口岸截获来自澳大利亚的丽蝇进行形态与DNA条形码鉴定,通过与澳大利亚的蝇类专家进行标本交流,进一步确认鉴定结果;并与相似种螳丽蝇进行形态与DNA条形码上的比较。结果通过形态观察与DNA条形码鉴定,所截获的丽蝇为杜比亚丽蝇;经科技查新,确认为中国未见分布种。结论 DNA条形码是传统形态分类的有力补充,对在国境卫生检验检疫工作中及时鉴定媒介生物,减少媒介生物本身及其携带病原体的扩散,减少对公共卫生的安全威胁起到重要作用。     

DNA条形码技术在河北省鼠疫疫源地鼠种鉴定中的应用

目的以河北省鼠疫疫源地常见鼠为对象,探讨DNA条形码技术在鼠类鉴定方面的可行性。方法2012年在河北省鼠疫疫源地内采集鼠肝脏标本,并保存整只鼠,制作剥制标本,提取基因组DNA,用通用引物PCR法扩增线粒体COⅠ基因,并测序。将测序结果与GenBank中其他鼠类物种的DNA条形码进行BLAST比对,并构建分子进化树。结果87份样本均能通过PCR扩增出特异性COⅠ基因条带,其中85份样本所构建的分子进化树结果与形态学鉴定结果一致,2份样本结果有差异,经反复鉴定头骨及形态标本,发现为现场鉴定错误。结论DNA条形码技术能够对鼠类进行有效的物种鉴定,同时还可以探讨啮齿动物属、种分类单元的系统发育问题。     

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。     

Linking morphometric and genetic divergence with host use in the tick complex,Ornithodoros capensis sensu lato

Host specific adaptations in parasites can lead to the divergence of conspecific populations. However, this divergence can be difficult to measure because morphological changes may not be expressed or because obvious changes may simply reflect phenotypic plasticity. Combining both genetic and phenotypic information can enable a better understanding of the divergence process and help identify the underlying selective forces, particularly in closely-related species groups. Here, we link genetic and morphometric data to understand divergence patterns within the Ornithodoros (Carios) capensis complex, a group of soft ticks (Argasidae) exploiting colonial seabirds across the globe. Species designations in this complex were historically based on larval morphology and geographic location. However, recent work has suggested that divergence within the group may be at least partially linked to host specificity. We therefore first examined population genetic structure of ticks in relation to host use and geography. These analyses revealed strong structure in relation to host use, both when populations were sympatric and widely allopatric, with a secondary effect of geography. They also demonstrated the presence of several novel and presumably undescribed species exploiting these seabird hosts. We then used geometric morphometrics (landmark and outline analyses) to test whether host-associated genetic divergence is always accompanied by the same phenotypic changes. We found that morphological variation (size and shape) correlated well with genetic structure; tick size and shape varied strongly in relation to host type, and weakly with geography. These results support the hypothesis that speciation in this tick group has been more strongly shaped by host use than by geographic barriers per se. The revealed phenetic patterns now require detailed investigation to link them with host-specific selective forces.     

用DNA条形码技术鉴定阿拉善黄鼠和达乌尔黄鼠

目的 探索阿拉善黄鼠和达乌尔黄鼠的分类鉴定方法。方法 运用DNA条形码技术测定两地黄鼠的线粒体细胞色素C氧化酶亚基Ⅰ（COⅠ）基因序列,分析遗传距离,构建系统发育NJ树。结果 11份阿拉善黄鼠的种内遗传距离〈2%,4份达乌尔黄鼠的种内遗传距离〈1%,两地黄鼠的遗传距离介于8%~9%之间,同时,NJ树将两地黄鼠分成2个独立分支。结论 阿拉善黄鼠为独立物种。DNA条形码技术可用于黄鼠种类鉴定。     

Laboratory colonization of Mansonia uniformis, Ma. indiana and Ma. bonneae in Malaysia

Methods are described for the laboratory colonization of Mansonia uniformis, Ma. indiana and Ma. bonneae in Malaysia. Gravid females oviposited in 500 ml beakers with a layer of water covered with small leaves of Salvinia. Newly hatched larvae were set up in a basal medium of guinea pig dung and water or liver powder, yeast powder and water. Larvae attached to aquatic plants or 'Keaykolour' ruffia snow white paper. The cultures with paper gave better yields than those with plants. Production of Ma. uniformis was higher than the other two species. Twelve generations of Ma. uniformis and 11 generations of Ma. indiana and Ma. bonneae were monitored in the laboratory.     

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。     

DNA barcoding of Iranian radicine freshwater snails begins to untangle the taxonomy and phylogeography of intermediate hosts of schistosomiasis and fasciolosis from the Middle East and across Central Asia

In the Middle East radicine snails are of considerable medical and veterinary importance acting as vectors of trematodes. In Iran, such snails are responsible for the transmission of the zoonotic trematodes Schistosoma turkestanicum and Fasciola gigantica. Historically, Radix gedrosiana has been incriminated as an important intermediate host for both trematodes, however, controversy remains over the snail’s true taxonomic status. This species has been determined using morphological characters that has resulted in erroneous identification of species, affecting understanding of population biology, and ultimately affecting vector incrimination. In this current study DNA barcoding using cox1 and phylogenetic analyses revealed that snails identified as R. gedrosiana from Iran split into two separate species, Radix euphratica and Ampullaceana sp. The cox1 also provided useful insights into the evolutionary history of R. euphratica populations. Phylogeographic analyses indicated that R. euphratica had an Iraqi/Iranian origin approximately 3.3 MYA and exists as a large stable population across the Middle East and Central Asia, and a lack of genetic differentiation between geographical isolates. Such molecular barcoding techniques are crucial for the identification of radicine snails of Iran being invaluable for the monitoring of zoonotic flukes, understanding the distribution of infection and the accurate incrimination of snail vectors.     

一例输入性板齿鼠的种类鉴定与分析

目的 对1份从入境集装箱中检获的鼠样品进行种类鉴定。方法 运用形态学方法及DNA条形码技术进行鉴定分析。结果 鼠样品体长约252 mm,尾长220 mm,后足长40 mm,毛色呈黑褐色,cytB基因序列与板齿鼠同源性最为接近,与不同板齿鼠的遗传距离为0.048~0.175。结论 经形态鉴定和DNA条形码比对,确定为板齿鼠,系浙江省口岸首次检获该鼠种,为加强外来医学媒介生物防控提供了基础依据。     

Relative abundance and blood feeding behavior of nocturnally active culicine mosquitoes in western Kenya

At 2 sites in western Kenya targeted for future malaria vaccine trials, adult culicine mosquitoes were sampled over one year by 5 collection techniques to assess human exposure to potential vectors of pathogens other than malaria. Collections included 20,910 females representing 19 species in Kisian and 4,312 females of 11 species in Saradidi. Common species in Kisian included Culex quinquefasciatus (71.4%), Mansonia uniformis (15.8%), Ma. africana (6.2%), Aedes mcintoshi (2.0%), Coquillettidia fuscopennata (1.9%) and Ae. ochraceus (1.8%). Common species in Saradidi included Cx. quinquefasciatus (92.7%), Cx. nebulosus (4.5%) and Ma. uniformis (1.0%). Human-bait collections identified 16 man-biting culicine species in Kisian and 9 in Saradidi. Man-biting rates at Kisian for the 5 most common species were 1.8, 14.6 and 13.5 times higher than at Saradidi for indoor, outdoor and tent collections, respectively. Exposure indoors was estimated to be 1,277 bites/man/year at Kisian and 720 at Saradidi. Blood meal identification for 1,083 mosquitoes confirmed that the common culicine species feed primarily on humans and cows.     

Wing shape divergence between Rhodnius prolixus from Cojedes (Venezuela) and Rhodnius robustus from Mérida (Venezuela).

The existence of Rhodnius robustus as a species distinct from Rhodnius prolixus has long been the main epidemiological question about Chagas disease transmission in Venezuela and surrounding countries. These two taxa are morphologically and genetically very similar, but only R. prolixus is assumed to colonize houses and transmit Chagas disease to humans. R. robustus is assumed to be an exclusively sylvatic species, restricted to palm trees. If robustus and prolixus are actually the same species, the theoretical possibility exists of sylvatic specimens invading houses, even after insecticide application, and a control strategy similar to that of the successful Southern Cone Initiative against Triatoma infestans would be difficult to consider. Since no valid alternative control strategy exists, the answer to this biological question could be decisive about the future of vector control in this region. Although we believe genetic techniques are best suited to define species boundaries, we present here an example of the relevance of modern morphometrics in dealing with such an issue. Using both traditional and geometric morphometrics, we compared the wing size and shape in both sexes of these two taxa reared in the same laboratory for one generation. R. robustus specimens were collected from palm trees in the state of Mérida (Venezuela), and R. prolixus were collected from houses in the state of Cojedes (Venezuela). Our study provided no argument to question their specific status. Even after one generation of living in the same laboratory conditions, the two lines showed clear size differences, divergent allometric trends, and significant allometry-free differences in shape. These results suggest that R. robustus (Mérida, Venezuela) and R. prolixus (Cojedes, Venezuela) are distinct evolutionary units. Due to the epidemiological importance of this question, further studies in other geographic areas of Venezuela are required to accurately define the relationships of R. robustus and R. prolixus.     

Mosquitoes of the mangrove forests of India: part six--Kundapur, Karnataka and Kannur, Kerala

Mosquitoes of 26 species belonging to 16 subgenera and 11 genera were recorded in the Kundapur mangroves of Karnataka, and 17 species belonging to 11 subgenera and 7 genera were recorded in the mangroves of Kannur, Kerala along the west coast of India. Genera recorded were Aedes, Anopheles, Armigeres, Culex, Heizmannia, Lutzia, Mansonia, Ochlerotatus, Tripteroides, Uranotaenia, and Verrallina. Species common to both mangrove forests were Ae. albopictus, Ae. vittatus, An. jamesi, Ar. subalbatus, Cx. gelidus, Cx. infantulus, Cx. pseudovishnui, Cx. sitiens, Cx. tritaeniorhynchus, Oc. wardi, Ur. atra, and Ve. luguhris. Tree holes and swamp pools were the common larval habitats, with more species occurring in tree holes in Kundapur than in Kannur. Adults of Ae. albopictus, Ae. vittatus, Ar. aureolineatus, Ar. subalbatus, Cx. bitaeniorhynchus, Cx. sitiens, Ma. uniformis, and Ve. lugubris bloodfed on humans.     

Relative potency of Bacillus thuringiensis var. israelensis and Bacillus sphaericus 2362 for Mansonia titillans and Mansonia dyari

Bacillus thuringiensis var. israelensis (B.t.i.), HD-968-S-1983, and a primary powder of Bacillus sphaericus 2362 were assayed against Mansonia titillans and Ma. dyari. The susceptibility of the 2 species to the individual bacterial toxins was similar. The B.t.i. LC50 values were 50.5 micrograms/liter for Ma. dyari and 54.7 micrograms/liter for Ma. titillans; the B. sphaericus LC50 values were 339.0 micrograms/liter and 347.2 micrograms/liter, respectively. The log dose-probit mortality lines were parallel for both target species. The potency of B. sphaericus relative to B.t.i. was 0.15 for Ma. dyari and 0.16 for Ma. titillans.     

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条形码在国境卫生检疫中的应用

综述了DAN条形码的发展历史、识别原理以及公共数据库,并讨论了DNA条形码在检验检疫领域的应用前景。DNA条形码与DNA芯片技术的结合,可在检验检疫领域中实现非专家鉴定。     

DNA barcoding of hard ticks (Ixodidae), notes on distribution of vector species and new faunal record for Croatia

Molecular methods are increasingly being utilized for accurate identification of ticks (Acari: Ixodidae), especially in cases of morphologically highly similar species. In this study, we performed molecular research of the tick fauna in Croatia using DNA barcoding method. Ticks were sampled in three biogeographical regions and thirteen species were recorded: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma marginatum, Ixodes frontalis, Ixodes hexagonus, Ixodes kaiseri, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus. Ixodes kaiseri is for the first time recorded in the fauna of Croatia. Of the thirteen hard tick species analyzed in this study, pathogens from different groups (bacteria, protozoa and viruses) have been detected in eight species in Croatia so far. For the important vector species R. sanguineus s.s., new distributional data for Croatia are given. The standard COI barcoding region was amplified, and the sequences were analyzed by species delimitation methods together with the sequences of conspecific and congeneric species from the public BOLD database. Our specimens of H. punctata represent a new, genetically distinct MOTU. A brief overview of the available public DNA barcoding data for Ixodidae is presented, highlighting the need for an integrative approach for the clarification of the taxonomic status of problematic Ixodid taxa. The results provide a basis for the establishment of a molecular data platform for the Ixodidae of the Croatian fauna.     

The wing venation patterns to identify single tsetse flies

This is the first study to explore the potential of various geometric morphometrics methods to help the morphological diagnostic of tsetse species, vectors of human and animal trypanosomiases in sub-Saharan Africa. We compared landmarks, semilandmarks and outlines techniques on male and female samples of species, and suggested adapted strategies according to the countries and their own Glossina fauna. We could compare up to 7 taxa belonging to the three main subgenera of the Glossina genus: Nemorhina (5 species), Glossina (1 species) and Austenina (1 species). Our sample included the major vectors of sleeping sickness: G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes and G. f. quanzensis, as well as two important vectors of African animal trypanosomoses: G. tachinoides and Glossina morsitans submorsitans. The average level of correct species recognition by the wing shape was satisfactory, and slightly higher for females than for males. The best scores of correct assignment, in both sexes, were obtained by the contour technique (96% of correct attribution in females, 92% in males), slightly higher than for semilandmarks (95% and 91%) or landmarks (94% and 89%) techniques. We made our images of wings freely available to be used as reference images (http://mome-clic.com), and we describe the conditions and the analytical steps to be followed to identify unknown specimens using external reference images. Under adequate conditions, such use of reference images obtained from a free access server could help species identification of new samples anywhere in Africa.