【作者】 云月利；

【导师】 雷朝亮； 彭宇；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 草间钻头蛛Hylyphantes graminicola是棉田中数量最多的蜘蛛。本论文首次进行了Wolbachia感染对蜘蛛生殖及适合度的影响研究,并通过草间钻头蛛的交配行为检测到Wolbachia可以通过交配方式进行传播,为以后进一步开展蜘蛛Wolbachia的研究提供了理论与实验依据。本论文的主要研究结果如下：1草间钻头蛛体内Wolbachia的感染检测及遗传多样性分析所检测的14个地理种群(新乡、菏泽、五指山、洛阳、高陵、陵水、西双版纳、武鸣、石家庄、太谷、南京、肥东、兴山、武汉)的草间钻头蛛均有Wolbachia感染,其中河南新乡草间钻头蛛Wolbachia的感染率最高,达到52.94%,山东菏泽的次之,为45.45%,Wolbachia感染率较低的草间钻头蛛主要分布在安徽肥东(8.62%)、湖北兴山(8.1%)和湖北武汉(7.69%)。对测序得到的7个地理种群共69个wsp基因片段用软件DnaSP4.10进行多态性位点分析,结果表明,总有效位点数565个,不变位点数380个,可变位点数185个。遗传距离分析显示同一地理种群Wolbachia品系的wsp基因遗传距离并不全都接近零或等于零,不同地理种群的Wolbachia品系之间也会出现遗传距离极小或为零。对7个地理种群Wolbachia wsp序列的单倍型进行遗传多样性分析结果显示,五指山(WZS)种群的单倍型多样度最高(0.90000),平均核苷酸差异度最大(83.70000),核苷酸多样性最高(0.45243)。AMOVA(Analysis of Molecular Variance)分子方差分析的结果表明,84.96%的遗传变异存在于种群内个体间,遗传分化是极显著的(Fst=0.15042,P<0.01)。2草间钻头蛛体内Wolbachia的多位点序列分型Wolbachia多位点序列分型结果显示,11个地理种群的Wolbachia品系分别属于10个不同的Wolbachia ST (sequence type),且这10个ST是Wolbachia MLST数据库中新出现的序列类型。其中洛阳(LY)和菏泽(HZ)具有完全相同的ST。Wolbachia系统发育分析(GarliML.MP和Bayesian分析)结果显示,8个ST(ST-177,ST-178,ST-179,ST-180,ST-181,ST-182,ST-183 and ST-184)属于Wolbachia B组,2个ST属于Wolbachia A组(ST185和ST186)。通过ARLEQUIN 3.1中的Mantel软件对不同地理种群草间钻头蛛COI遗传距离和相应的Wolbachia两两遗传距离的相关性分析结果显示,Wolbachia品系的两两遗传距离与宿主的COI遗传距离间相关性显著(r2=0.394,p=0.013,p<0.05)。而宿主的地理距离与Wolbachia品系遗传距离之间相关性分析结果显示二者没有明显的相关性(r2=0.027,p=0.665,p>0.05),宿主地理距离与宿主的线粒体COI遗传距离相关性分析显示二者相关性不显著(r2=0.027,p=0.135,p>0.05)。3 Wolbachia对草间钻头蛛生殖及适合度的影响对草间钻头蛛4种交配方式的研究结果表明,♀+×(?)-、♀-×(?)+、♀+×(?)+和♀-×(?)-4种不同交配形式的各个交配对都能正常地交配和产卵(♀+表示感染Wolbachia的雌蛛；(?)-表示未感染的雄蛛；♀-代表未感染的雌蛛；(?)+代表感染的雄蛛),而且产的卵都能正常发育,即♀-×(?)+之间没有发生细胞质不亲合性。对感染与未感染Wolbachia的草间钻头蛛产卵量、卵历期和卵孵化个体数的统计分析结果显示,感染与未感染Wolbachia的草间钻头蛛第一个卵袋卵孵化历期有显著差异(P<0.01)。两种方法去除草间钻头蛛体内Wolbachia的结果显示,浓度为1 Omg/ml四环素盐酸盐的水溶液饲喂草间钻头蛛50d后,40%的草间钻头蛛去除了Wolbachia共生菌。浓度为3.75mg/ml的草间钻头蛛人工饲料喂养草间钻头蛛50d后,50%的被处理蜘蛛个体去除了体内共生菌Wolbachia。4 Wolbachia水平传递方式的研究未感染Wolbachia的草间钻头蛛捕食感染有Wolbachia的果蝇,Wolbachia感染检测结果表明：捕食后2h、12h、24h、48h、72h的草间钻头蛛均未检测出Wolbachia感染。以感染Wolbachia的单雌系果蝇为饲料,把草间钻头蛛从二龄喂养至成熟,50头草间钻头蛛的Wolbachia感染检测结果均显示未感染。这一结果表明草间钻头蛛通过捕食Wolbachia阳性的果蝇不可能获得Wolbachia感染。水稻田节肢动物群落Wolbachia的感染检测结果也未获得关于捕食传播的生态学及分子生物学证据。通过设置两组草间钻头蛛交配对♀+×(?)-、♀-×(?)+,Wolbachia交配传播研究的结果显示,通过宿主的交配行为,Wolbachia既可由感染的雌性草间钻头蛛传递给未感染的雄性草间钻头蛛,也可由感染的雄性传递给未感染的雌性。更多还原

【Abstract】 Hylyphantes graminicola is one of the most prevalent spiders in the cotton field. In this study, the effect of Wolbachia on host’s reproduction and fittness were analysed, and the routine for Wolbachia horizontal transmission were also conducted. We firstly detected that CI(cytoplasmis incompatibility) was not happened in H. graminicola, and Wolbachia can be transmitted by mating of H. graminicola in this study. All these results will provide useful theoretical and empirical foundation for later studys. The followings are the main results of this study.1 Wolbachia infection and genetic divergence analyses of Wolbachia wsp gene in different Hylyphantes graminicola populationsThe result of Wolbachia detecting in 14 geogrophical populations (XX, HZ, WZS, LY, GL, LS, BN, WM, SJZ, TG, NJ, FD, XS, WH) of Hylyphantes graminicola indicated that Wolbachia infection existed in all 14 populations of H. graminicola. The infection frequency of XX (Henan Province) popuation was the highest (52.94%), then the HZ (Shandong Province) population (45.45%). The lower infection frequency of Wolbachia existed in FD population (8.62%), XS population (8.1%) and WH population (7.69%).The result for DNA polymorphic sites analyses of Wolbachia wsp gene (69 sequences from 7 populations) by DnaSP4.10 indicated that total number of sites was 560. Invariable (monomorphic) sites was 380, and variable (polymorphic) sites was 185. The result of genetic distance analyses showed that genetic distances within a same population of H, graminicola were not always near or equal to 0, and genetic distances of Wolbachia wsp gene between different populations will be equal to 0 sometimes. The result of wsp gene haplotype showed that the haplotype diversity of WZS population was the highest (0.90000). The mean number of pairwise differences of WZS population was the highest (83.70000), and the the nucleotide diversity of WZS population was the highest (0.45243). Through AMOVA analyses,84.96% genetic variation happened within population, and the genetic differentiation within popultion was so significant (Fst=0.15042, P<0.01).2 Wolbachia MLST typing of Hylyphantes graminicola A total of 10 STs, new to the MLST database, were found in 11 H. graminicola geographic populations, and the STs from LY and HZ are identical. The 10 STs were assigned to supergroup A and supergroup B respectively based on the phylogenies of concatenated MLST data. Based on the ML, MP and BI analyses, eight STs (ST-177, ST-178, ST-179, ST-180, ST-181, ST-182, ST-183 and ST-184) were assigned to supergroup B, and two STs (ST185 and ST 186) to supergroup A.The result of Mantel test indicated the correlation was significant between pairwise distance of H. graminicola COI and associated Wolbachia MLST concatenated data (r2=0.394,p=0.013,p<0.05). We also checked the correlation between Wolbachia MLST concatenated data and host’s geographic distance, and the result showed that no correlation was found between Wolbachia divergence and host’s geographic distance (r2=0.027,p=0.665, p>0.05). No correlation was found between pairwise distance of H. graminicola COI and its geographic distance (r2=0.027,p=0.135, p>0.05).3 The effect of Wolbachia on the reproductive and fittness of H. graminicolaThe result shows that 4 types of mating pairs in H. graminicola((?)+×(?)-、(?)-×(?)+(?)+×(?)+和(?)-×(?)-) all have normal mating behavior and can produce valid egg sacs, and CI was not found within mating pair(?)-×(?)+. Through analysing the number of egg sacs, developmental duration of eggs and decendents of each egg sacs between Wolbachia positive H. graminicola and Wolbachia negative H. graminicola, the significance was found in developmental duration of the first eggs sacs(P<0.01).The result of Wolbachia removing from its host showed that 40% Wolbachia positive H. graminicola lost Wolbachia through feeding on l0mg/ml antibiotics solution for 50 days, and 50% Wolbachia positive H. graminicola also lost Wolbachia through feeding on 3.75mg/ml antibiotics artificial feed (the density of antibiotics in artificial feed) for 50 days.4 Wolbachia horizontal transmission Through Wolbachia negative H. graminicola predating Wolbachia positive Drosophila melangastor, the result of Wolbachia detecting showed that no Wolbachia infection was detected in H. graminicola (who have predated Wolbachia positive Drosophila melangastor).Two mating groups between Wolbachia positive individuals ((?)+or (?)+) and negative individuals (()?-or(?)-) were assigned in this study, and the molecular evidence was detected by sequencing wsp (Wolbachia surface protein) gene fragments. The results shows that through mating, positive spiders ((?)+or(?)+) can transmit Wolbachia to negative spiders ((?) or(?)-), and this is the first report about Wolbachia horizontal transfer through mating behavior in intraspecific level.更多还原