【作者】 熊焰；

【导师】 洪晓月；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2007， 硕士

【摘要】 桔小实蝇(Bactrocera dorsalis Hendel，或Dacus dorsalis Hendel)和红火蚁(Solenopsis invicta Buren)是2种世界性检疫害虫。桔小实蝇现已扩散到北美洲、大洋洲和亚洲的20多个国家和地区；目前在中国分布于台湾、广东、广西、湖南、贵州、福建、海南、云南、四川等省区；该虫以幼虫蛀食为害柑桔、柚子、桃、莲雾、榴莲、番石榴、芒果、香蕉、荔枝、龙眼等数百种栽培作物和野生植物，传播途径隐蔽，是当今为害最为严重的农业害虫之一，在我国被列为进境植物检疫二类危险性害虫。红火蚁原分布于南美洲的Parana流域，目前已入侵美国、巴西、秘鲁、玻利维亚、阿根廷、乌拉圭、波多黎哥、澳大利亚、新西兰、马来西亚、安提瓜岛和巴布达岛、巴哈马群岛、特立尼达和多巴哥等国家和地区，并在世界范围内呈现出迅速扩散趋势；在中国台湾、香港、澳门、广东、广西和湖南等省区有分布；农业部已将其定为中华人民共和国进境植物检疫性有害生物和全国植物检疫性有害生物。红火蚁危害人类健康，破坏生态系统和公共设施，影响农业生产并耗费巨额防治费用。江苏省面临着上述两类害虫严峻的入侵趋势，对这两类有害生物的防控手段以进出口贸易检疫为主。本文拟分别基于该两种有害生物的生物学和生态学特征，应用地理信息系统(Arc GIS8.1)对其在江苏省的入侵风险进行分析。此外，还尝试应用分子生物学的方法，以mtDNA COI基因为分子遗传标记，研究火蚁属的种间关系和中国的5个红火蚁地理种群的遗传差异。这将有助于我们从宏观和微观两个方面认识有害生物，了解其入侵途径和爆发机制，为有效地控制其扩散和危害奠定理论基础。一、桔小实蝇在江苏省的入侵风险分析温度是决定桔小实蝇地理分布和扩散趋势的最重要的关键因子。基于桔小实蝇的生物学和生态学特性，在Excel平台上统计分析了江苏省13个站点20年的气象数据。数据分析表明江苏省是桔小实蝇的潜在适生分布区；有效积温分析表明，桔小实蝇一年内在江苏省可发生5-7代；基于地理信息系统Arc GIS 8.1的桔小实蝇地理分布预测图表明其潜在的高发期为7月下旬和8月上旬；预测结果还显示，桔小实蝇在常州、溧阳、苏州、南京和吕泗等31°N-32°N地区有相对较好的适生性。二、红火蚁在江苏省的入侵风险分析红火蚁的适生情况取决于某一地区蚁后是否能够产生一定临界数量有翅蚁，而低温导致过低的生殖率和越冬死亡则证明红火蚁不适合在该地区生存繁衍。虽然海拔高度和降雨量可能对红火蚁的分布与扩散能产生影响，但是在分析了江苏的农业地理概况和江苏省13个站点20年的气象数据后，海拔高度和降雨量不会限制其在江苏省的分布与扩散。数据分析表明江苏省是红火蚁的潜在适生分布区；有效积温分析表明，红火蚁一年内在江苏省可发生1-3代；基于地理信息系统Arc GIS 8.1的红火蚁地理分布预测图表明其潜在的高发期为7月下旬和8月上旬；预测结果还显示，红火蚁在常州、溧阳、苏州、南京和吕泗等31°N-32°N地区有有较高的入侵风险。三、中国红火蚁种群分子遗传结构的初步研究分子遗传标记己经被国内外众多学者广泛应用于昆虫学的各个领域，如分类鉴定、系统分化、害虫防治、生态适应等。目前分子生物学技术在外来有害生物入侵风险研究中主要应用于有害生物的诊断、近缘种的分类鉴定和群体间系统发育分析两个方面。建立在丰富的核酸序列信息的分析基础之上的分子种群系统发育分析，有助于更深入地了解有害生物种间关系和种群的遗传变异、起源、进化和入侵、定殖及扩散等问题。线粒体细胞色素氧化酶亚基Ⅰ(mtDNA COI)基因适合用来分析亲缘关系较近的种、种下分类阶元以及地理种群之间的系统关系。本研究采用DNA序列分析法，对采自中国不同地区红火蚁的mtDNA COI基因序列进行比较，分析并揭示红火蚁与其相似种的关系及红火蚁各地理种群间的遗传差异。试验用红火蚁于2005-2006年采集于广东吴川市、广西北流市、广西陆川市、广西岑溪市和湖南张家界市，保存于100％乙醇中。采用UNIQ-10柱式动物基因组DNA抽提试剂盒提取红火蚁的基因组DNA。应用合适的引物对其进行PCR扩增，历经PCR产物回收纯化、连接和转化后用扩增引物做测序引物。经PCR检测后将阳性克隆用于测序。每个地理种群随机抽取至少3个样本进行测序，以其一致序列为准。最终成功的扩增出长度为965 bp的mtDNA COI中央序列。将获得的DNA序列先提交到GenBank数据库中，然后利用NCBI站点“BLAST”工具进行序列分析、DNA序列检索。并引入阿根廷12个地区和巴西8个地区的红火蚁mtDNA COI序列，以阿根廷和巴西的火蚁属其它8个种mtDNA COI序列作为外群，用GenDoc软件进行序列同源性比较；用BioEdit软件进行序列编辑；用Clustal X软件进行序列比对；比对结果输入MEGA 2.1软件，用邻接法构建系统发育树。研究结果发现，利用mtDNA COI序列作为分子遗传标记可以较好的解决红火蚁与其近似种的区分问题。火蚁属其它种如S. pusillignis、S. electea、S. daguerrei、S.megergates、S. macdonaghi、S. interrupta作为外群较先以极高的置信度被划分出来，与红火蚁S. invicta分别处于不同的进化分支上。中国的红火蚁种群在进化上被分为两个分支，但是它们都和阿根廷的红火蚁亲缘关系较近。红火蚁广西陆川种群、广西北流种群和湖南张家界种群的亲缘关系较近，和阿根廷一部分种群处于同一支内；而由于红火蚁广西岑溪种群和广东吴川种群的序列完全相同，它们和阿根延红火蚁另外一些地理种群处于一个较大的分支内。红火蚁传入中国的历史不长，分子进化水平上的差异不会相差太大，因此我们推测中国红火蚁很可能起源于阿根廷或其他南美地区，但是进入的时间和途经可能有别。5个地理种群间遗传分化程度总体而言比较低，其原因一方面可能与我们推测该虫在中国的历史较短有关。由于该虫在中国发展的时间相对较短，估计还不足以积累更多的遗传变异，各种群仍然还保持着相对密切的遗传联系，故表现为遗传分化程度不高；另一方面，这5个种群遗传分化的程度较低也可能与各种群所在区域内的生态条件基本一致有关。群体的分化程度主要是对所在的生态条件相适应的结果，如果其所产生环境作用强度及方向大体相同，则各分布区内的种群将难以形成显著的分化。更多还原

【Abstract】 The oriental fruit fly Bactrocera dorsalis Hendel and the red imported fire ant Solenopsisinvicta Buren are two important quarantine pests worldwide. It is believed that B. dorsalishas expanded to occupy more than 20 countries and districts of North America, Oceaniaand Asia and it also has been widely distributed in the provinces of Taiwan, Guangxi,Fujian, Hainan, Hunan, Guizhou, Yunan, Sichuan and Jiangsu of China at present. Withquite indiscoverable carrying pathway, the larva B. dorsalis causes harm to hundreds ofcrops and wild plants such as orange, pomelo, peach, wax apple, durian, guava, mango,banana, lichee, longan and so on, which has become one of the most serious pestsnowadays. S. invicta, an original pest in Parana drainage area, has invaded America, Brazil,Peru, Bolivia, Argentina, Uruguay, Porto Rico, Australia, Malaysia, Antigua, Bahamas,Trinidad and Tobago, and showed quick diffusion tendency worldwide. It is proved that S.invicta has established new populations in Taiwan, Hong Kong, Macao, Guangdong,Guangxi and Hunan in China. S. invicta endangers people’s health, havocs ecosystem andpublic utilities, creates great economic lost to Agriculture, and consumes vast sum ofmoney for prevention. Both the two species are ranked in the national and internationalquarantine pests list. Quarantine measurements are mainly adopted for preventing anyaccidental introduction of these two pests to China. It is obvious that Jiangsu Province, thewindow of east China, is facing a great invasive threat by the two pests. In order to find outthe invasive risk of the pests in Jiangsu, we try to apply Arc GIS 8.1 in our study basing onthe biological and ecological characteristics of B. dorsalis and S. invicta. Furthermore, wechoose S. invicta as a model and mitochondrial DNA cytochrome subunit I (mtDNA COI)gene as a genetic maker to examine the interspecific relationship between Solenopsisspecies and the genetic variation of five geographic populations of S. invicta from China atthe molecular level. Our studying result helps people for recognizing the pests’ invasionpathways and the break-out mechanisms using both macro and micro visions and aims tolay a consolidated theoretical basis for preventing the further diffusion and detrimentultimately. 1. The risk analysis of invasive B. dorsalis in Jiangsu Province of ChinaIt is known that the temperature is the most important key factor for regulating colonymetabolism and activity, which is therefore used as the primary ecological factor fordetermining the geographic distribution and diffusion tendency of B. dorsalis. Based on thebiological and ecological characteristics of the oriental fruit fly, B. dorsalis, we analyzedthe meteorological data in twenty years from 13 sites in Jiangsu Province using MicrosoftExcel system. The result shows that Jiangsu Province is a potential suitable establishmentarea for the pest. Effective accumulated temperature analysis indicates that the pest mayhave five to seven generations a year in the province. The geographical distribution mapforecasted via the Geographic Information System (Arc GIS 8.1) indicates that regions suchas Changzhou, Liyang, Suzhou, Nanjing and Lvsi, whose altitude fall in 31°N～32°N,show relatively better suitability. The map also indicates that the pest can establish itspopulation or have outbreaks easily from late July to early August.2. The risk analysis of invasive S. invicta in Jiangsu Province of ChinaLifetime female alate production of red imported fire ant, S. invicta is calculated so asto determine whether the colony can exist and develop itself or not. The fact that extremelow temperature will cause lower reproductive rates and winter kill, which gives evidencethat the target area is not suitable for geographic distribution and diffusion. Though heightabove sea level and precipitation amount will affect its suitability to some extent, we deemthat the above two parameters will not limit the geographic distribution and diffusion of S.invicta in Jiangsu Province after the analysis of meteorological data in twenty years from13 sites in the Province. The result shows that Jiangsu Province is a potential suitableestablishment area for the pest. Effective accumulated temperature analysis indicates thatthe pest may have one to three generations a year in the province. The geographicaldistribution map forecasted via the Geographic Information System (ArcGIS 8.1) indicatesthat regions such as Changzhou, Liyang, Suzhou, Nanjing and Lvsi, whose altitude fall in31°N～32°N, show relatively better suitability. The map also indicates that the pest canestablish its population or have outbreaks easily from late July to early August. Quarantineofficials should be vigilant for any accidental introductions of this pest in the susceptibleregions and time.3. The molecular genetic structure of S. invicta populations from China Molecular genetic marker technology has been wildly applied in such entomologicaldomains like classification& determination, systematic evolution, ecological adaptation andpest control, etc. The application of molecular technology presently in Pest Risk Analysis(PRA) are mainly two aspects, classification& determination at individually level andevolution analysis at phylogenetic level. The population phylogeny analysis based on themass information of nucleic sequence gives an insight into the reorganizations of not onlythe interspecific relationship but also the intraspecific heredity & variation, origination,evolution, invasion, establishment, expansion, etc. As mtDNA COI gene is recognized as asuitable genetic marker for analyzing the systematic relationship of the relative species, thesubspecific categories and the geographic populations, in our study we use moleculartechniques to examine the interspecific relationship between Solenopsis species and theintraspecific variation of mtDNA COI sequences among the populations of S. invicta.Samples for the experiment were collected from Wuchuan of Guangdong Province,Beiliu, Cenxi, Luchuan of Guangxi Province, and Zhangjiajie of Hunan Province. GenomeDNA of S. invicta was extracted by using UNIQ-10 Column-shaped Extraction Kit. PartialDNA sequence of mtDNA COI for each population was then PCR amplified using one pairspecial primers. The purified PCR products were subsequently connected with pGEM-Tvector and transformed to susceptive Escherichia coli. After PCR test to the transformantsusing the same primers, the positive clone was screened out for sequencing. The partialmtDNA COI of each geographic population for S. invicta was finally determined by theidentical sequences after three clones at least were randomly sampled for sequencing. Apartial mtDNA COI sequence of 965 bp length was proved to be successfully amplified foreach population in our study. The DNA sequences were then submitted to the GenBankdatabase and then compared online with the published sequences by similarity searchengines such as BLAST in NCBI website then calculated in GeneDoc and aligned usingClustalⅩcomputer program. Twenty foreign mtDNA COI sequences of S. invicta and eightmtDNA COI sequences of Solenosis species as out-group, previously published by otherresearchers, were introduced to our research for the construction of phylogenetic tree.Analysis of genetic and phylogenetic relationship was performed by using MEGA2.1.Genetic relationships among every geographical population were estimated based on thepair-wise matrix of sequence divergences by Kimura-2 Parameter method. Phylogenetictree was constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ treewere assessed by bootstrapping from 1000 pseudo-replications. We come to a conclusion that the discrimination between the red imported fire ant, S.invicta, and other allied species of Solenopsis can be resolved preferably by using mtDNACOI gene as a molecular genetic marker. We could see from the phylogenetic tree that mostspecies in Solenopsis as an out-group can be separated distinctly from the S. invicta at veryhigh confidence levels and they are located in distinct clades in the tree. The populations ofS. invicta from China ares close with the Argentine populations based on the analysis tomtDNA COI, no matter they are divided into two major clades. Populations from Luchuanand Beiliu of Guangxi Province and Zhangjiajie of Hunan Province are quite relative andthey are in a major clade with some Argentine populations; Populations from Cenxi ofGuangxi Province and Wuchuan of Guangdong based on the analysis to mtDNA COI arecompletely the same and they are in another major clade with some other Argentine S.invicta populations.The introduction history of the exotic pest, S. invicta in China is not too long, whichresults in the delicate molecular evolutional discrepancy between the different geographicpopulations in China. We argue that the Chinese S. invicta are most probablely originatedfrom Argentina accidentally, however the introduction pathway and time maybe differentfrom each other. Generally speaking, the genetic variation between the populations of S.invicta in China is low relatively. We deem that one reason is the short history of the pest inChina, which results in insufficient variation accumulation and lower variation rate, so thepopulations of S. invicta still keeps a close genetic relationship with each other; anotherreason is that the approximate ecological conditions of their habitats bring about the lowerselection pressure. Population variation level sometimes relies on how much the organismsadapt themselves to the environment. If the evolutionary directions and intensities causedby environment are approximately the same, there will not be a distinct variation betweenthe populations in an area.更多还原