【作者】 李志国；

【导师】 时连根； 苏松坤；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2014， 博士

【摘要】 近年来,全球部分地区蜂群数量的减少,尤其是2006年冬季至2007年春季,美国发生的大范围的蜜蜂突然消失现象——蜂群崩溃失调症(colony collapse disorder, CCD),类似的情况也发生在欧洲的部分地区,引发了严重的授粉危机。初步的研究表明以色列急性麻痹病毒(Israeli acute paralysis virus, IAPV)很可能是CCD的致病因素,尽管对于CCD的病因依然并未明确,现在普遍认为蜜蜂的健康主要受到蜂螨、病毒、蜜蜂微孢子虫、杀虫剂残留及营养不良等因素的影响,这些因素之间的交互作用或许是近年来世界范围内蜂群损失的原因。本研究调查了IAPV等常见蜜蜂病毒在浙江省地区的流行情况,描述了中、意蜂病害类别的差异进而分析了中、意蜂等与免疫相关基因的表达差异；通过分子检测发现了IAPV等蜜蜂病毒另一新的传播载体——蜜蜂球囊菌,揭示了IAPV等蜜蜂病毒新的生物学特征；基于蜜蜂伸吻反应(proboscis extension response, PER)行为实验及射频识别(radio frequency identification, RFID)技术,研究了IAPV对花粉采集蜂的梯度蔗糖溶液反应性及归巢能力的影响；初步研究了东方蜜蜂微孢子虫、蜂螨协同侵染对蜜蜂体内IAPV滴度及相关免疫基因表达的影响。主要结果如下：1.浙江省常见蜜蜂病毒的流行情况及中、意蜂病害类别的比较及其基因表达差异从浙江省七个地区的38个意蜂蜂场采集蜜蜂样品用于常见六种蜜蜂病毒的检测。IAPV,蜜蜂残翅病毒(Deformed wing virus, DWV)的检测率最高,分别为29%、27%；克什米尔蜜蜂病毒(Kashmir bee virus, KBV),囊状幼虫病毒(Sacbrood virus, SBV)的检测率为5%；慢性蜜蜂麻痹病毒(Chronic bee paralysis virus, CBPV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus, ABPV)的检测率分别为4%、3%。病毒的组织嗜性调查发现,相较于头部,爬蜂及死蜂的胸部、腹部被病毒感染的可能性更大。通过对141个意大利蜜蜂(Apis mellifera L.)蜂场的调查,发现80%的蜂场存在不同程度的蜂螨侵染；21%的蜂场发现白垩病；18%的蜂场遭受了因农药中毒而引发的蜂群损失事件；6%的蜂场存在幼虫腐臭病；5%的蜂场发生了不明原因的蜂群消失事件。与意蜂蜂群相比,通过对39个中华蜜蜂(Apis cerana cerana)蜂场的调查,发现中蜂蜂场不存在蜂群消失事件且不存在蜂螨寄生蜜蜂的问题；近90%的蜂场存在不同程度地被大蜡螟危害的现象。鉴于上述中华蜜蜂、意大利蜜蜂生物学及病害类别方面的差异,我们通过高通量测序技术分析了中、意蜂头部基因的差异表达情况,发现了约2,370个与代谢、免疫、应激反应相关的差异表达基因。中、意蜂之间编码抗菌肽基因的差异表达表明了中、意蜂在抵御病害方面存在的分子机制差异。2.首次在蜜蜂球囊菌中检测到IAPV等蜜蜂病毒及病毒反义链蜜蜂球囊菌(Ascosphaera apis, A. apis)是蜜蜂的一种真菌性病原,可以感染蜜蜂幼虫并引起白垩病。已有研究表明真菌病毒存在于所有的主要真菌门类。本研究从白垩病虫尸中培养、分离蜜蜂球囊菌。通过RT-PCR检测所收集的球囊菌菌丝中常见7种蜜蜂病毒的存在情况。我们发现IAPV、黑蜂王台病毒(Black queen cell virus, BQCV)及DWV这三种病毒可以在球囊菌丝中被检测出,此外,这些病毒对应的反义链亦可以被检测出,说明球囊菌可能是IAPV、BQCV及DWV新的传播载体,这一发现揭示了蜜蜂病毒的一个新的生物学特征。系统进化分析发现源于蜜蜂、球囊菌的病毒序列形成了个单源的进化关系,但形成两个很明显的不同的进化簇。区别于蜜蜂病毒序列,来自于球囊菌的病毒基因序列组成了一个很明显的谱系。3. IAPV感染影响蜜蜂的蔗糖反应性行为及蜜蜂的归巢能力本研究通过蜜蜂PER行为和RFID技术两个方面来研究IAPV对意大利蜜蜂采集行为及归巢能力的影响。来源于健康蜂群(无可检测水平的蜜蜂病毒感染)的花粉采集蜂被人为地注射IAPV以诱导病毒感染的状态。结果表明,与未被IAPV感染的蜜蜂相比,被IAPV感染的蜜蜂对低浓度的糖水反应性更强。两天后,在被IAPV感染的蜜蜂头部可检测到大约107拷贝的病毒粒子。此外,与未被IAPV感染的蜜蜂相比,被IAPV感染的蜜蜂的归巢能力显著降低(p<0.05)。然而,IAPV感染不影响蜜蜂的致敏化反应及习惯化反应。实验数据表明IAPV在蜜蜂头部的感染或许使得IAPV能够让蜜蜂的采集行为发生紊乱并扰乱蜜蜂大脑负责学习、导航及定位的功能,结果使得蜜蜂对蔗糖溶液的反应阈值降低并在归巢的途中迷失。4.东方蜜蜂微孢子虫、蜂螨共同侵染蜜蜂对蜜蜂体内IAPV滴度及相关免疫基因表达影响的初步研究蜜蜂微孢子虫(Nosema ceranae)、蜂螨(Varroa destructor)对蜜蜂的协同侵染可以影响蜜蜂体内IAPV滴度及蜜蜂抗菌肽、Eater、卵黄蛋白原等免疫相关基因的表达,但免疫基因表达的变化趋势不是很明显,有的甚至呈相反的表达趋势,这些初步的数据显示蜜蜂微孢子虫、蜂螨、IAPV及蜜蜂之间相互作用关系的复杂性。更多还原

【Abstract】 The honey bee colony losses across parts of the world in recent years and that honey bee colonies disappeared abruptly in the US and parts of Europe from winter2006to spring2007have triggered pollination crisis. The sudden collapse of honeybee colonies is known as colony collapse disorder (CCD) and was initially thought to be caused by Israeli acute paralysis virus (IAPV). Following more research, it is generally accepted that a variety of factors including Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition may cause CCD. Besides, interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years.Viral prevalence of common honey bee viruses including IAPV, Deformed wing virus (DWV), Chronic bee paralysis virus (CBPV), Acute bee paralysis virus (ABPV), Sacbrood virus (SBV) and Kashmir bee virus (KBV) was surveyed in parts of Zhejiang province, and comparisons between Apis mellifera and Apis cerana for pathogen diversity and differential expression of immune genes were furthered analyzed in the study; first detection of honey bee viruses including IAPV, Black queen cell virus (BQCV) and DWV and their replication in the fungi Ascosphaera apis (A. apis) uncovers a new biological feature of honey bee viruses; effects of IAPV on sucrose responsiveness and homing ability of pollen foragers were investigated based on proboscis extension response (PER) and radio frequency identification (RFID) techniques; synergistic effects of two pathogens including Varroa mites and Nosema ceranae on IAPV titer dynamics and expression of immune genes in honey bees were also studied. These results were summarized as follows:1. A survey of six honey bee viruses in Zhejiang province and comparisons between Apis mellifera and Apis cerana for pathogen diversity and molecular mechanisms underlying the differential response to pathogensSamples collected from38typical apiaries located in7different regions of Zhejiang province were tested for presence of six common honey bee viruses. The most prevalent virus was IAPV, with the detection rate of29%in all samples, followed by DWV (27%), KBV (5%), SBV (5%), CBPV (4%) and ABPV (3%). Examination of tissue tropism of the viruses showed that thorax and abdomen of crawling bees and dead bees were more likely to be positive for viruses.A regional survey covering honey bee viruses, mite and wax moth infestation, pesticide poisoning, foulbrood, chalkbrood, which are detrimental to the health of colonies was carried out in the study. Of141apiary locations consisting of colonies of Apis mellifera ligustica (Aml),5%of apiaries suffered from inexplicable colony losses. Honey bee colonies from about80%of apiaries were observed with different levels of Varroa mite infestation.21%of apiaries had chalkbrood, and6%of apiaries had foulbrood.18%of apiaries underwent colony losses resulting from pesticide poisoning.39apiaries raising A. cerana cerana (Acc) did not confront with colony mortality or Varroa mites compared with A. mellifera. Nearly90%of Acc apiaries were more or less infected with the greater wax moth (Galleria mellonella), followed by foulbrood positive apiaries (18%).Given the differences in biology and pathogen diversity between Acc and Aml, we first used the Illumina-Solexa deep sequencing technology to describe the differences in the heads of Acc and Aml foragers at the gene expression level, and found about2,370differentially expressed genes related to metabolism, immune and stress response between Acc and Aml. The differential expression of antimicrobial peptide genes suggested that the defense mechanisms against pathogenic microbes in Acc and Aml are different from each other. That may be a reason why there were no reports about the unusual mortality of eastern honeybee (Acc) colonies in comparison to the drastic loss of western honeybee (A. mellifera) that has been occurring worldwide in recent years.2. First detection of honey bee viruses and their replication in the fungi Ascosphaera apisThe Ascosphaera apis (A. apis) is a fungal pathogen of honey bees causing chalkbrood in honey bee larvae and it has been shown that fungal viruses or mycoviruses have been found to be widespread in all major fungal taxa. Therefore, the chalkbrood mummies from which the A. apis was cultured and isolated, and the mycelium of A. apis were further used for detecting the common honey bee viruses in the present study. We demonstrated that honey bee viruses including DWV, Black queen cell virus (BQCV) and IAPV could infect and replicate in the fungal pathogen A. apis that causes honey bee chalkbrood disease, revealing a novel biological feature of honey bee viruses. Replication of honey bee viruses in A. apis indicated that A. apis may act as a possible vector during honey bee viruses transmission.The phylogenetic analysis showed that phylogenetic relationships between viruses of fungal and honey bee origins are all monophyletic, forming two clusters in the phylogenetic trees distinctly. Viruses from A. apis constitute a distinctive lineage, separated from the clades of viruses identified in honey bees. Further studies are warranted to investigate the impact of the viruses on the fitness of their fungal host and phenotypic effects the virus-fungus combination has on honey bee hosts.3. Viral infection affects sucrose responsiveness and homing ability of forager honey bees, Apis mellifera L.In the present study, the effects of a honey bee virus, IAPV, on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on PER assays and RFID systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around107copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. However, sensitization test and habituation test, two patterns of nonassociative learning, showed no significant difference between foragers infected with IAPV and foragers infected with PBS. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.4. Effects of Varroa mites and Nosema ceranae on IAPV titer dynamics and expression of immune genes in honey beesIAPV titer dynamics and expression of Abaecin, Eater and Vitellogenin (Vg) in honey bees can be affected by co-infection with Varroa mites and Nosema ceranae. However, no obvious trend regarding expression of immune genes was found in the study. Some immune genes showed trends opposite the expect trends. This preliminary study indicated that the complicated interactions beween Nosema ceranae、mites、IAPV and honey bees. Further study is needed regarding the complicated interactions.更多还原