【作者】 刘钦来；

【导师】 文继舫； 蔡继峰；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 背景法医昆虫学在实践中主要应用于死亡时间(postmortem inteval, PMI)推断。法医昆虫学推断PMI的原理主要有两种：一是研究尸体上主要嗜尸性昆虫的生长发育规律来推断PMI；二是研究尸体上昆虫的种类及演替规律来推断PMI。即依据蝇类的生活史、群落组成随季节和时间的变动规律来推断PMI。中国幅员辽阔,不同地域昆虫的种类及生活习性均不相同,不能简单地照搬其它地方的研究资料来进行法医实践,但有必要借鉴前人的研究方法来对各地区嗜尸性昆虫群落演替展开调查研究,发现当地的优势物种,并加以饲养,进一步研究其生长发育规律,逐步建立适合于当地的嗜尸性昆虫种类的数据库和推测系统,为法医实践提供依据。对嗜尸性昆虫,尤其是嗜尸性丽蝇进行快速、准确的种类鉴定,是PMI推断的关键环节,有着特别重要的作用。丽蝇往往是第一批到达尸体并产卵的嗜尸性昆虫,具有重要的法医学意义,但一些近缘丽蝇物种和丽蝇幼虫之间的鉴别尚缺乏明显的形态学特征。分子生物学物种鉴定技术,是形态学鉴定的有力补充,此类方法快捷、简便、有效。中国地理环境多样,丽蝇种类繁多,还需要积累更多的基因资源来进行鉴定研究。因此有必要在全国范围收集大量丽蝇样本,并开发新的嗜尸性丽蝇的分子标记,完善嗜尸性丽蝇的鉴定系统。同时完善各种嗜尸性蝇类的发育历期、生长曲线、及嗜尸性昆虫的演替等资料。但目前,国内这些方面的资料还很缺乏。目的1.在全国多个地区收集丽蝇样本,对我国常见嗜尸性丽蝇的种类、分布及嗜尸特性展开调查,并在长沙地区夏季猪尸体上对常见嗜尸性昆虫群落种类和演替规律进行调查研究。2.利用278bp COI线粒体基因片段和272bp COI片段对来自全国28个地区的130个丽蝇样本进行种类鉴定,检测这两个基因片段对不同丽蝇物种的鉴别效力,构建并完善我国嗜尸性丽蝇的分子标记系统。3.饲养优势嗜尸性蝇种,并研究其生长发育规律,为本地区法医昆虫学实践奠定理论基础和提供科学依据。方法1.在全国不同经纬度的28个地区,用动物尸体诱捕丽蝇并收集样本。夏季在长沙地区连续三年用猪尸体进行演替实验。2.用Primer5.0设计引物,用匀浆法、蛋白酶K+酚／氯仿／异戊醇法提取足够的基因组DNA,用Promega试剂盒扩增PCR,用常规琼脂糖凝胶电泳检测DNA和PCR产物。扩增产物用胶回收试剂盒纯化,产物批量送测序。测序结果用Chromas及ClustalW等软件阅读、比对,截取等长度片段。用Mega4.0进行序列分析及系统发育树的构建。将所获得嗜尸性丽蝇基因片段的数据上传GenBank数据库。3.夏季在室内自然条件下饲养丝光绿蝇、大头金蝇和棕尾别麻蝇,测量它们的幼虫的体长和体重,还测量蝇蛹的长、宽、重,并绘制它们随时间变化的生长曲线图、构建相关方程。结果1.三年以来在全国范围17个省28个地区收集了大量丽蝇样本,初步形态学鉴定并选出13种130个丽蝇样本。2.连续三年夏季在长沙地区野外及室内用猪尸体进行演替实验,发现嗜尸性昆虫29种,分属3目15科。3.用278bp COI和272bp COI基因片段对来自全国多个地区的丽蝇进行物种鉴定,发现这两个片段均适合于嗜尸性丽蝇的物种鉴定,尤其是一些近缘种类的鉴别。4.用278bp COI片段进行鉴定时,发现丝光绿蝇Lucilia sericata (Meigen,1826)和叉叶绿蝇Lucilia caesar (Linnaeus,1758)存在种内基因地域差异。而272bp COI片段进行鉴定时,发现大头金蝇Chrysomya megacephala (Fabricius,1794)和新陆原伏蝇Protophormia terraenovae (Robineau-Desvoidy,1830)存在种内基因地域差异。5.成功地饲养了丝光绿蝇L. sericata、大头金蝇C. megacephala和棕尾别麻蝇Boerttcherisca peregrine (Robineau-Desvordy,1830)。绘制了幼虫体长、体重,蝇蛹长、宽、重随时间变化的生长曲线图。发现幼虫体长和时间的关系吻合三次方程L=aT3+bT2蝇蛹体重则符合一次方程W=aT+b。相关性显著(R2>0.9,P<0.05)其中,L代表蛆长,W代表蛹重,T代表时间,a、b、c、d为常数。结论1.在我国多个地区进行嗜尸性丽蝇样本的采集,补充了嗜尸性丽蝇种类及分布资料。2.三年来在长沙地区进行了嗜尸性昆虫演替实验,对推断PMI等法医实践具有一定的实用价值。3.短片段COI(278bp COI和272bp COI基因片段),具有快速、简便、经济、有效鉴别物种等优点,适合基层法医工作者应用。4.向GenBank数据库上传了13种丽蝇物种的145条COI基因片段,完善了数据库并初步构建了我国嗜尸性丽蝇分子标记系统。5.饲养一些有法医学重要意义的嗜尸性苍蝇并进行生长发育规律研究,获得了一些基础性科学数据,为法医实践工作提供了理论依据,也为今后研究多种嗜尸性蝇类的生长发育规律提供了方法。更多还原

【Abstract】 BackgroundIn practical applications, forensic entomology is mainly used for the determination of the postmortem interval (PMI). There are two main ways to infer the PMI:Firstly, to study the growth and development laws of the sarcosaphagous insects on the corpses with the PMI. Secondly, to study the insects that are on the corpses and their corresponding succession to provide important clues for inferring the PMI. In different regions, the insect species and their living habits are not the same. China has a vast territory, and the entomological evidence from one region can not be used for the forensic practice in other regions directly. Therefore, creating local databases with related ecological data for the identification of sarcosaphagous insects is strongly recommended. Additionally, it is important to expand the investigations of various regions based on the previous research experience, to investigate the sarcosaphagous insect community succession, to find the dominant species of local regions and to study their growth and development laws. At that point we can establish local database and estimation system suitable for sarcosaphagous insects collected from different sites of Changsha. The rapid and accurate identification of sarcosaphagous insect species, especially the sarcosaphagous calliphorids, is a crucial step for estimation of the PMI. Calliphorids have important forensic significance, because they are usually the first colonizers, and then spawn and breed the next generations on the corpses. The obvious morphological characteristics to differentiate some closely related blowfly species or blowfly larvaes are still lacking. As a strong complement to the morphological identification method, molecular identification technique is a fast, easy, and effective method. Due to the diverse geographical environment in China, Calliphorids have a wide range of species, so more genetic resources still need to be collected for identification studies. It is necessary to collect calliphorid samples nationwide, and find some new molecular markers for calliphorid identification. Meanwhile, it is important to perfect the developmental duration tables and the growth curves of the sarcosaphagous flies as well as the succession data of the sarcosaphagous insects. However, such data are still scarce in China.Objectives1. To collect the calliphorid samples in multiple regions of China, and to investigate the species and distribution of some common calliphorids. To study the insect communities and their succession laws on the pig’s corpses in the region of Changsha in summer,.2. To identify the species of130calliphorids collected from28regions in China by278bp COI gene fragment and272bp COI fragment. To detect the ability of two gene fragments to identify some types of blowfly species. To set up and perfect the molecular marker system for Calliphoridae in China.3. To raise three dominant fly species and study their growth and development laws. To lay the foundation for local practice of forensic entomology.Methods1. Animal corpses are used to trap calliphorids in28regions of China in different latitudes and longitudes. Putting pig corpses at selected outdoor and indoor sites for succession experiment in Changsha in summer for three years.2. The primers for amplification of short COI fragment are designed by Primer5program, mtDNA of all samples are extracted by improved DNA extraction method for grinding tissues or Proteinase K+phenol/chloroform/isoamyl alcohol method, and mtDNA is amplified by the Promega PCR Kit. Conventional agarose gel electrophoresis is used for the detection of DNA and PCR products. Amplification products are purified by gel extraction kit and the purified products are analyzed by dideoxy-chain termination method of Sanger. Sequencing results are aligned by Chromas and clustering analysis is performed by ClustalW software. Finally, the sequences are analyzed by Mega4.0and phylogenetic tree is constructed. Gene data of sarcosaphagous calliphorids collected from different geographic regions and climate conditions are uploaded to GenBank database for accumulating Chinese calliphorid genetic data.3. L. sericata, C. Megacephala and B. peregrine are raised under the room-temperature in Changsha in summer. The body length and weight of the larvae, the length, width and weight of the pupae are measured, and the growth curves over the time are established.Results1. Calliphorids were trapped from28regions across17provinces in the country within three years, samples were obtained and preliminary morphological identification was executed.130samples of13calliphorid species were selected.2. Succession experiments were carried out at selected outdoor and indoor sites in Changsha in summer for three years.29sarcosaphagous species were found on the pig corpses belonging to three orders and15families respectively.3.278bp COI fragment and272bp COI fragment were used for the identification of all kinds of calliphoridae species collected all over the country. These results show that the fragments are suitable for identification of the sarcosaphagous calliphoridae species, especially some closely related calliphorid species.4. When the278bp COI fragments were used for identification, the intraspecific geographical differences were discovered in species L. sericata (Meigen,1826) and L. Caesar (Linnaeus,1758). And when272bp COI fragments were used for identification, the intraspecific geographical differences were discovered in species Chrysomya megacephala (Fabricius,1794) and Protophormia terraenovae (Robineau-Desvoidy,1830).5. Three sarcosaphagous fly species were successfully raised, i.e. L. sericata, C. megacephala and Boerttcherisca peregrine (Robineau-Desvordy,1830), and the growth charts of the larval body length and weight, fly pupae length, width and weight changes over the time were drawn. The results indicate that the larval body length and time relations coincide with cubic equation L=aT+bT2+cT+d and fly pupa weight is in line with the simple equation W=aT+b. The correlation was a significant difference (R2>0.9>,P<0.05).(L represents the length of the maggots, W represents the weight of the pupal, T represents the time, a, b, c and d represent constants).Conclusion1. A number of calliphorid samples are obtained in various districts around the country. The data concerning species and distribution of calliphorid flies are enriched.2. The sucession experiments have been carried out in Changsha region for three years, which will have practical forensic significance for the PMI estimation.3. Short fragments of COI (278bp and272bp) have the advantage of being fast, simple, economic and effective in identification of species. They are suitable for grassroot forensic workers.4. A large number of COI fragments have been uploaded into the GenBank database. The molecular maker system of calliphorid flies has been preliminarily set up in China.5. Some forensically important sarcosaphagous fly species have been raised, and a preliminary study has been done into the growth and development laws about them. Some basic and scientific data have been obtained. They will serve as theoretical basis for the forensic work. They will also provide methods for the future research of the growth and development laws of the sarcosaphagous flies.更多还原