狄斯瓦螨和蜜蜂残翅病毒对蜜蜂健康的协同影响

世界各地大范围的西方蜜蜂Apis mellifera蜂群损失现象已引起科学界和公众的持续关注。狄斯瓦螨Varroa destructor和蜜蜂残翅病毒(Deformed wing virus,DWV)是西方蜜蜂群中最主要的两大生物威胁。尽管二者侵害蜜蜂均已有较长历史,但直至近十年来的研究才发现两者间的协同效应对蜂群健康的影响远超过其单独作用时所造成的危害:(1)蜜蜂残翅病毒可在狄斯瓦螨体内大量复制,继而进一步传播;(2)狄斯瓦螨的刺吸行为使病毒粒子跨越寄主的生理屏障而直接进入蜜蜂血淋巴;(3)狄斯瓦螨的寄生促使蜜蜂残翅病毒的高毒力毒株在蜂群中优势扩增和盛行;(4)狄斯瓦螨影响蜜蜂个体发育与免疫系统等生理机能,以致降低了蜂群对病毒的抵抗力;(5)蜜蜂残翅病毒对宿主造成的免疫抑制有利于狄斯瓦螨的寄生与繁殖。狄斯瓦螨、蜜蜂残翅病毒和西方蜜蜂间的关系已经成为昆虫外寄生物、病原体与寄主相互作用研究的一个典型模型。本文对近十年该领域的相关研究进行综述,以期为蜂群损失的原因调查以及昆虫寄生虫、病原微生物与寄主间关系的研究提供参考和借鉴。     

蜜蜂残翅病研究进展

蜜蜂残翅病病毒(Deformed Wing Virus, DWV)是蜜蜂残翅病的病原体,能够侵染各个发育阶段的蜜蜂,严重时导致蜂蛹发育缓慢,成蜂翅膀畸形、体型萎缩、行动乏力,蜂群早衰;不严重的隐形感染也会缩短蜜蜂寿命。DWV借助于狄斯瓦螨Varroa destructor在全球广泛传播,并产生DWV-B和DWV-C等变异体,它们的共同作用促使蜂群群势急剧衰弱,是导致"越冬蜂群的消失(OCL)"和"蜂群崩溃失调症(CCD)"的主要病因之一,给养蜂业造成巨大损失,而目前尚没有特异性的有效治疗药物。DWV的自身特征、与狄斯瓦螨的关系、以及单独或者和狄斯瓦螨共同作用对蜜蜂的影响等研究都是开发防治技术方法的基础,本文从生物学、流行学、病理学和防治技术等方面对残翅病病毒的最新研究成果进行了综述。     

安徽省七种蜜蜂病毒的发生与流行研究

【目的】调查安徽省内7种常见蜜蜂病毒:蜜蜂畸翅病毒(Deformed wing virus,DWV)、以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus,ABPV)、慢性麻痹病毒(Chronic bee paralysis virus,CBPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、囊状幼虫病病毒(Sacbrood virus,SBV)、克什米尔病毒(Kashmir bee virus,KBV)的感染发生情况,为安徽养蜂业可持续健康发展提供理论依据。【方法】运用反转录RT-PCR和序列分析比对的方法对安徽省内21个乡镇中的38个蜂场蜜蜂样品进行研究分析,以获得以上7种蜜蜂病毒的特异性发生情况。【结果】意大利蜜蜂Apis mellifera蜂场感染率:DWV(64%),IAPV(43%),CBPV(32%),ABPV(14%),BQCV(11%);中华蜜蜂Apis cerana蜂场感染率:DWV(80%),IAPV(40%),CBPV(30%),ABPV(10%),BQCV(0)。SBV和KBV在所有的蜜蜂样品中均未检测到。【结论】DWV,IAPV,CBPV,ABPV,BQCV在安徽省内大范围都存在发生流行现象,SBV和KBV对安徽蜜蜂的潜在危害可能性小。     

外寄生诱导的寄主蜜蜂免疫和病理学反应

了解外寄生物一病原体一寄主三者之间的关系,不仅有重要的理论意义也有广阔的应用前景。狄斯瓦螨（Varroa）是蜜蜂的外寄生螨,可以导致蜜蜂后代的大量死亡,同时也可以携带和诱导很多病毒的发生。蜜蜂蛹感染狄斯瓦螨后,残翅病毒（deformed wings virus,DWV）的扩增水平明显增加,     

蜜蜂残翼病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立

蜜蜂残翅病毒(Deform wing virus,DWV)已成为世界上最著名、分布最广、研究最为深入的昆虫病原.虽然DWV以前存在于蜜蜂种群中,但一种新的媒介一外寄生螨Varroa破坏体的到来和全球传播,极大地促进了 DWV的流行病学.为了建立快速、准确且能定量分析的蜜蜂残翅病毒,DWV检测方法,本研究参照GenBank中已登录的DWV高度保守的3C-RdRp基因区域,设计了 1对特异性引物和带有羧基荧光素(Fast Auxiliary Memory,FAM)与淬灭基团(Eclipse)标记的TaqMan探针,并对反应条件进行优化,建立了检测DWV的TaqMan-MGB探针荧光定量RT-PCR检测方法(TaqMan qRT-PCR),同时对该检测方法的特异性、敏感性、重复性进行了试验,并对临床样品进行检测.结果显示:该方法最佳上下游引物和探针浓度分别为12.5μmol/L和10μmol/L,最佳退火温度为59℃;敏感性试验中,对DWV质粒标准品检测下限为2.58×101拷贝/μL;本方法特异性较好,对蜜蜂常见病毒-蜜蜂慢性麻痹病毒(Chronic bee paralysis virus,CBPV)、蜜蜂急性麻痹病毒(Acute paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、囊状幼虫病毒(Sacbrood virus,SBV)、以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)和中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)无交叉反应;且批内变异系数和批间变异系数均小于2％.利用该方法对49份临床疑似样本进行检测,其中DWV阳性样本为35份,检出率高于常规RT-PCR方法,且病毒载量大于1×107拷贝/只样本数共24份,表明辽宁和河北部分地区感染DWV蜂群中病毒载量较高,流行情况比较严重.因此,本研究建立的TaqMan探针荧光定量RT-PCR方法具有灵敏度高、特异性强、重复性好等特点,能够用于DWV病原监测、流行病学调查和病毒定量分析等相关研究.     

狄斯瓦螨和蜜蜂病毒对意大利蜜蜂蜂群越冬的影响

越冬期是蜂群损失最主要的阶段.通过比较分析45个意大利蜜蜂Apis mellifera ligustica蜂群在繁殖越冬蜂前的狄斯瓦螨Varroa destructor寄生率和病毒感染情况、越冬表现及越冬期存活蜂群的病毒感染情况等,探究与越冬期蜜蜂健康紧密相关的影响因素.结果表明,繁殖越冬蜂前蜂群的狄斯瓦螨寄生率与蜜蜂残翅病毒(DWV)和以色列急性麻痹病毒(IAPV)基因组拷贝数呈中等线性相关关系(pDWV=0.003,pIAPV=0.001),且狄斯瓦螨寄生率低于9％的蜂群与DWV感染程度相关性更高,而寄生率高于9％的蜂群与IAPV的相关性更高.越冬期死亡蜂群在繁殖越冬蜂前的狄斯瓦螨寄生率和IAPV病毒基因组拷贝数均显著高于存活蜂群.狄斯瓦螨和IAPV是本次实验中意蜂蜂群越冬期健康的首要影响因素.结合狄斯瓦螨寄生率和IAPV基因组拷贝数的正相关性,本研究认为在繁殖越冬蜂前将蜂螨寄生率控制在较低水平(9％以下)能有效减少越冬期意蜂蜂群损失.     

蜜蜂羽化温度与卷翅病毒动态复制相关性研究

病毒复制动态变化对于病毒病的研究有着重要意义。选用蜜蜂卷翅病毒(deformed wing virus,以下简称DWV)作为研究模型,检测DWV隐性感染蜂群在不同温度条件下羽化工蜂体内的病毒浓度,以了解不适温度压力这一非生物因子对蜜蜂羽化率及蜜蜂体内DWV病毒复制动态的影响。实验结果显示不适羽化温度不仅会明显降低蜜蜂羽化成功率,而且新羽化的带毒蜜蜂体内DWV病毒拷贝量增加极显著。部分解释了冬季和早春低温环境下因病毒病暴发造成人工饲养蜂群非正常损失的原因。     

北京地区六种蜜蜂病毒病的流行病学研究

【目的】对蜜蜂的6种病毒:以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)、残翅病毒(Deformed wing virus,DWV)、囊状幼虫病病毒(Sacbrood virus,SBV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、慢性麻痹病毒(Chronic bee paralysis virus,CBPV)在北京地区的流行情况进行调查,以期为该地区蜜蜂病毒病的防控提供一定的理论依据。【方法】应用多重RT-PCR法确定上述6种病毒在该地区的感染情况,并通过序列分析确定特异性。【结果】在所有检测样本中均未检测到急性麻痹病病毒和慢性麻痹病病毒,感染率最高的是以色列急性麻痹病毒,其次是残翅病毒。检测的样本普遍存在混合感染。【结论】以色列急性麻痹病毒、残翅病毒、囊状幼虫病病毒、黑蜂王台病毒4种病毒可能在北京地区广泛分布。     

中华蜜蜂半乳糖凝集素AcGalectin的表达、纯化及性质分析

【目的】本研究旨在明确中华蜜蜂Apis cerana cerana半乳糖凝集素(galectin)的功能及其与病毒的相互作用。【方法】提取中华蜜蜂的总RNA,利用RT-PCR技术获得Galectin基因,然后利用生物信息学软件对基因序列及其推导氨基酸序列和结构特征进行分析;再依据大肠杆菌Escherichia coli密码子的偏嗜性对该基因密码子进行优化、原核表达,并制备重组蛋白;最后,通过Far-western blotting技术检测纯化的重组蛋白与纯化的中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)、慢性蜜蜂麻痹病毒(Chronic bee paralysis virus,CBPV)和残翅病毒(Deformed wing virus,DWV)的结合情况。【结果】从中华蜜蜂中克隆到Galectin基因,并将其命名为Ac Galectin(Gen Bank登录号:MG557559),其cDNA全长为1 473 bp。Ac Galectin空间结构比较稳定,包含一个半乳糖识别结构域。系统进化树表明,Galectin明显分为两簇,膜翅目蜜蜂科昆虫的Galectin形成一个簇,而其他昆虫Galectin形成另一簇。经密码子优化后的Ac Galectin基因能够在大肠杆菌感受态细胞BL21(DE3)中高效表达。纯化的重组Ac Galectin蛋白可与CSBV和CBPV结合,但不与DWV结合。【结论】结果表明Ac Galectin与蜂病毒CSBV和CBPV有结合作用。本研究为Ac Galectin在CSBV和CBPV感染过程中的功能研究奠定了基础。     

狄斯瓦螨与蜜蜂的寄生关系

蜜蜂Apis spp.是重要的经济昆虫,也是研究最为深入和广泛的社会性昆虫。狄斯瓦螨Varroa destructor是蜜蜂的体外寄生虫,是对西方蜜蜂Apis mellifera蜂群健康危害最为严重的生物因子。近十多年来,由于全球蜂群损失现象严重,而同时以蜜蜂为代表的授粉昆虫的健康直接关系到农业生产和生态安全,关于狄斯瓦螨与蜜蜂的寄生关系的研究受到极大关注。作为社会性昆虫的一种体外寄生虫,狄斯瓦螨不仅需要适应蜜蜂个体的生长发育,借助蜜蜂化蛹期间的封盖期完成繁殖,同时还要能够逃避蜜蜂社会性行为的清理。本文从狄斯瓦螨的生物学特性以及其与寄主间的互作关系等方面进行论述,以期进一步理解寄生虫-无脊椎动物寄主(特别是社会性昆虫寄主)的相互作用,为寄生虫病学研究提供借鉴。     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.