不同活性的东方蜜蜂微孢子虫（Nosema cerana）诱导西方蜜蜂（Apis mellifera）应答反应及免疫信号通路的激活研究

As a model organism, the honey bee is the most important pollinator and plays a vital role in the ecological balance, agricultural development and science research. In the last dacade, large honey bee losses have occurred globally (CCD) and bring out.disastrous effect in the apiculture and related areas. While the causes for these declines are not fully understood. .Nosema was thought be one of the main factors of CCD. The treatment of Nosema was based on chemicalcan so far and the bio-controlling for Nosema was still no studied. In our preliminary data, we firstly show that autoclaved N. ceranae spores fed to A. mellifera larvae reared in vitro (vaccination) and subsequently fed live Nosema spores as adults (inoculation) have infection levels reduced adult death(85%). The proposed research will firstly explore the best immunization system for Apis mellifera to resistant Nosema cerana, based on the effect of vaccine different stage vaccine on the rate, account, survival and immunization-related genes expression. Then we will investigate the midgut transcriptome profile of the lavae or newly emerged bees of Apis mellifera that infected with autoclaved or alive Nosema based on the Illumina Genome Analyzer platform. At the same time, we will measure the transcriptional level of genes involved in signal identification and signal transduction invlolved in innate immunity post infection with autoclaved or alive Nosema based on the real time qPCR. This presented proposal will help us to understand the efficacy of this vaccine during larval stage, or in newly emerged bees, and in field colonies. If successful, we will have developed a new treatment against N. ceranae and a transformative approach to treating the other honey bee diseases, and it will be useful in the close species, such as the bomble bees.

作为模式动物，蜜蜂是世界上最重要的授粉昆虫，在生态平衡、农业发展和科学研究等领域中起着至关重要的作用。近年来，全球蜂群数量急剧下降（CCD），给养蜂业及相关领域带来了灾难性的影响。蜜蜂微孢子虫被认为CCD的主要致病因素之一，目前主要依赖化学药物治疗。本课题前期研究首次发现饲喂灭活微孢子虫能显著降低蜜蜂感染微孢子虫病，并降低85%的死亡率。在此基础上，本项目将首先探索在不同阶段饲喂灭活微孢子虫对西方蜜蜂感染微孢子虫病概率、数量、寿命和相关免疫基因表达的影响，找出蜜蜂的最佳防御条件。然后建立微孢子虫感染蜜蜂的细胞系，对微孢子虫在细胞内的增殖进行观察研究。最后将利用illumina基因组测序分析平台对蜜蜂最佳防御条件下感染微孢子虫后的蜜蜂中肠表达谱数据进行分析；同时，利用RT-qPCR等技术测定防御与否条件下，蜜蜂感染微孢子虫后的免疫系统中参与信号识别和信号转导的相关蛋白的基因转录水平。

本项目首先研究了饲喂灭活微孢子虫对西方蜜蜂后期感染微孢子虫病的影响，结果发现：在微孢子虫感染概率方面，IP-0组（1日龄饲喂4万个灭活微孢子虫）与对照组0-0之间没有差异，因为它们后期一直都未被感染(Steel-Dwass Z=0.06, p=1.0)；0-40组（只在7日龄饲喂4万个新鲜提取的活包子虫）比0-0组感染率显著增加(Steel-Dwass Z=18.51, p<0.0001)；IP-40组（1日龄免疫饲喂4万个灭活微孢子虫、7日龄再次感染4万个新鲜提取的活微孢子虫）的感染率为58.77%，比 0-40组的65.45%少34%，差异极显著 (Steel-Dwass Z=-3.34, p<0.005)。饲喂微孢子虫组(IP-40、0-40和IP-0)的蜜蜂寿命显著低于对照组（0-0) (L-R Chi-square=22.39, 3 df, p<0.001)。其中，IP-0组比对照组少活0.97天(Chi-square=5.01, 1 df, p=0.025)； 0-40组比0-0少0.79天（Chi-square=5.40, 1 df, p=0.020）； IP-40组比0-0少1.82天(Chi-square=11.67, 1 df, p=0.0006)。4个免疫基因在各处理组之间的变化较大。在18日龄以前，Hym. 和Def.基因在IP-0中呈现显著的增加趋势；Api.基因也有增加趋势，但未形成显著的差异；18日龄以后即开始采集时，Api.、Hym.和Def.基因则由于微孢子虫感染导致表达显著受到抑制。然后利用illumina基因组测序分析平台对蜜蜂最佳防御条件下感染微孢子虫后的蜜蜂中肠表达谱数据进行分析。研究结果表明：Aba.、Api.、Hym., 和Def. 4个基因的表达水平呈现如下规律：在第7天和第21天时，IP-0处理组中Hymenoptaecin, 和Defensin的表达显著增加（P<0.01）。我们注意到Api.也有类似的趋势，但差异不显著(P>0.05)。随着蜜蜂年龄的增长和开始为采集做准备，Api., Hym.和 Def. 也都上调了，而接受致病孢子的处理由于感染而经历了表达抑制。对于Abaecin，我们观察到在第7天、和高压灭菌孢子的表达水平最高。在第21天，Aba.是四个基因中唯一一个在IP-0处理中没有明显高表达水平的基因。