【作者】 刘宝军；

【导师】 张星耀；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2014， 博士

【摘要】 随着全球化进程的加快和人类干扰导致生态系统完整性的破坏，生物入侵的风险不断加大，入侵种的扩散和暴发趋势严重，给入侵地的生态系统和国土安全造成巨大的灾害性损失。松材线虫是国际上公认的检疫性有害生物，给包括中国在内的很多国家造成了极为严重的经济、生态和社会损失。松材线虫侵染松树后，能在松树体内短时间内大量繁殖，并引起松树快速死亡。繁殖的生物学和生态学特征探讨，对掌握其高繁殖力机制、生态适应机制，及在机制探讨基础上提出基于繁殖控制的特异性、针对性的松材线虫的防治思路与技术，均具有重要意义，也可为松材线虫的致病机理和入侵机制的研究提供理论基础。本研究重点开展了松材线虫的择偶行为及其生态机制，并针对其择偶偏向开展了转录组学分析。主要结果如下：通过长时间的显微观察和录像回放的方法，研究了松材线虫的交配过程，并将松材线虫的交配过程定义和划分为搜寻、接触、交尾和逗留四个阶段，不同阶段的持续时长不同，四个阶段的时长分别为21.8±2.0min,28.0±1.9min,23.6±0.7min和7.2±0.5min。在交配过程中存在着具有典型特征化的动作，分别将其明确和定义为：逡巡、靠近、相遇、触摸、卷曲、定位、固定、射精、解尾、静止和徘徊等系列分步行为；松材线虫在交配过程中存在着明显的性吸引特征，存在着明显的同性内的竞争行为和异性间的选择行为，但雌性间的竞争行为相对较弱，而雄性间竞争的竞争强度明显大，雌性对配偶的选择行为典型而明显，但雄性的选择行为不明显。研究了一雌一雄（1♀+1♂）、多雌一雄（3♀+1♂）、一雌多雄（1♀+3♂）和多雌多雄（3♀+3♂）体系下松材线虫的择偶特征，发现不同体系下松材线虫总交配过程、各个交配阶段的时长、择偶时长、身体接触次数、首次接触交配成功率等都存在着明显的差异。四种体系下的交配效率测定实验结果表明，多雌一雄体系下的交配效率为189.6±2.1，显著的高于一雌多雄体系下的交配效率（110.0±0.8），同时多雌一雄体系下第一代后代的性比和成幼比分别为3.56±0.06和0.53±0.01显著的高于其他几种体系下的性比和成幼比，后代的交配效率更高。通过模拟交配效率曲线可知在雌雄比为3.4:1时，松材线虫的交配效率最高，并且偏雌性比下的交配效率要高于偏雄性比下的交配效率。松材线虫的交配对策符合状态制约选择理论，即偏雌性比下，线虫采用Y交配对策，交配效率高，后代的性比和成幼比较高，种群繁殖快，偏雄性比下，线虫采用X交配对策，交配效率低，后代的性比和成幼比较低，种群繁殖慢。研究结果还表明，松材线虫的雄性能够通过调整竞争强度来适应雌性的选择强度。开展了处女雌虫、处子雄虫、已婚雌虫和已婚雄虫的择偶偏向实验及交配效率测定，结果表明处女雌虫和已婚雌虫都会优先选择与处子雄虫进行交配，而处子雄虫、已婚雄虫对处女雌虫和已婚雌虫的择偶偏向性不明显。交配效率测定结果表明无论是处女雌虫还是已婚雌虫与处子雄虫交配效率（153.2±3.9、139.8±2.8）要显著的高于与已婚雄虫的交配效率（122.8±3.2、98.8±3.6），并且与处子雄虫交配的交配效率分别提高了24.8%和41.5%。同时雌虫与处子雄虫交配后获得的后代的性比（3.2±0.07、2.6±0.05）和成幼比（0.45±0.01、0.41±0.01）要显著高于与已婚雄虫交配的后代性比（2.7±0.04、2.2±0.04）和成幼比（0.32±0.01、0.31±0.01），因此与处子雄虫交配的后代的交配效率更高。开展了处女雌虫在面对潜在配偶时的转录组测序分析，结果表明，处女雌虫在面对潜在配偶时单独表达的基因有849个，与处女雌虫单独存在时相比，差异表达的基因有575个，这些差异表达的基因功能主要与运动、刺激响应、生长、发育、死亡、生殖、繁殖、代谢、定位、转录调控、翻译调控、抗氧化、细胞器等相关。发现了其它线虫上已知的69个与择偶交配相关的基因。通过对差异基因功能GO注释进一步筛选了与松材线虫刺激响应、繁殖、定位和运动相关的基因26个。这些都将为研究该类基因及其pathway途径对线虫择偶的影响，从而最终从分子水平上阐释松材线虫雌虫的择偶机制奠定了基础。更多还原

【Abstract】 With the acceleration of globalization and human disturbance in the ecological integrity ofthe system destruction, a large number of existing invasive species diffusion and outbreak ofserious risk of biological invasion has caused great disaster losses to the ecological system andhomeland security. Pine wood nematode (Bursaphelenchus xylophilus), as an agent of pine wiltdisease and the important quarantine target of many countries around the world, has beencaused many countries including china extremely serious economic, ecological and social loss.After infected a new tree, the PWN is propagated quickly in a short time within the stem ofpine trees and eventually death. The exploring of reproduction biology and ecologycharacterristics had important meaning for studing the high fecundity, ecology adaptationmechanism and providing a novel method to control pine wilt disease in future. It also couldprovide the theoretical basis of pathogesis and invasion mechanism.The study of mating choicebehavior of pine wood nematode, ecological mechanism and transcriptome sequencing ofmating preference were carried out. The main results are as follows.The mating behavior features of B. xylophilus by long-time microscope video andrepeated observation in one male and one female system. The mating behavior of B. xylophiluscould be decomposed into a series of sub-behaviors, including cruising, approaching,encountering, touching, hooping, locating, fixing, ejaculating, unfixing, quiescence androaming in sequence. According to the process of mating behavior, it could be divided into4different stages, i.e. searching, contacting, copulating and lingering stage. Time for differentstage varied significantly with21.8±2.0min,28.0±1.9min,23.6±0.7min and7.2±0.5min forfour mentioned stages respectively on average. Attraction between different sexes wasobviously observed, indicating that sexual pheromone might be involved in mating behavior ofB. xylophilus. In addition, mating choice of female to male was clearly observed. When onefemale was mixed with three males, male-male competition was obvious, which might be caused by the mating-choice pressure from female. Furthermore, intra-sexual competition offemale was also observed, but not obviously.Study on the choice characteristic of pine wood nematode in one male and one femalesystem (1♀+1♂), multiple females and one male system (3♀+1♂), one female and multiplemales system (1♀+3♂), multiple females and multiple males system (3♀+3♂). There weresignificantly differences in the whole mating process, the duration of different stages, durationof mating choice, contacting times and mating success rate for the first contact. The results ofmating efficiency of four systems showed that the mating efficiency in multiple females andone male system was189.6±2.1, significantly higher than that of one female and multiplemales system (110±0.8). Meanwhile, the sex ratio of offspring and ratio of adult to offspring inmultiple female and one male system were all higher than those of other systems. The offspringhad higher mating efficiency and may be more conducive to the breeding of the population.According to simulating the mating efficiency curves, the mating efficiency of pine woodnematode is the highest when the sexual ratio (female and male) is3.4:1, and the matingefficiency of female biased is higher than that of male biased. Mating strategy of pine woodnematode is accordance with status-dependent selection theory (SDS). In female biased system,Y mating strategy is used, the mating efficiency is high, sex ratio of offspring and ratio of adultto offspring is relatively high, the population is conducive to propagate quickly. In male biasedsystem, X mating strategy is used, the mating efficiency is low, sex ratio of offspring and ratioof adult to offspring is relatively low, the population is not conducive to propagate quickly.The results also showed that males adapted the intensity of competition according to thefemale’s selection intensity.Mating selection bias experiment and mating efficiency of virgin female, virgin male,mated female and mated male were determined. The results showed virgin female and matedfemale would preferred to mate with virgin male, but the mate preference of virgin male andmated male were not obvious. The results of mating efficiency determination showed themating efficiency of virgin or mated female when mated with virgin male were obviouslyhigher than that of mated with mated male and they increased24.8%and41.5%separately when the virgin female and mated female mated with virgin male. Meanwhile, the sex ratio ofoffspring and ratio of adult to offspring were significantly higher than those of mating withmated male. The mating efficiency of offspring was higher and more conducive to the breedingof population.Transcriptome sequencing was conducted when one virgin female encountered apotentialmate. The results showed that849genes were expressed individually when virgin female facedpotential mate,575genes were differentially expressed compared with virgin female existedalone. These functions of differentially expressed gene mainly included movement, stimulationresponse, reproduction, growth, development, death, metabolism, positioning, regulationl oftranscriptionand translation, antioxidant and organelles. Besides,69genes related to mating inother worms were selected.26genes were screened by gene function GO annotation whichrelated to stimulus response, breeding, positioning, and sports. The female mating choicemechanism will be elucidated by studying the influence of these genes and pathways on themating selection at the molecular level.更多还原