Energetic stress in the honeybee Apis mellifera from Nosema ceranae infection

Parasites are dependent on their hosts for energy to reproduce and can exert a significant nutritional stress on them. Energetic demand placed on the host is especially high in cases where the parasite-host complex is less co-evolved. The higher virulence of the newly discovered honeybee pathogen, Nosema ceranae, which causes a higher mortality in its new host Apis mellifera, might be based on a similar mechanism. Using Proboscis Extension Response and feeding experiments, we show that bees infected with N. ceranae have a higher hunger level that leads to a lower survival. Significantly, we also demonstrate that the survival of infected bees fed ad libitum is not different from that of uninfected bees. These results demonstrate that energetic stress is the probable cause of the shortened life span observed in infected bees. We argue that energetic stress can lead to the precocious and risky foraging observed in Nosema infected bees and discuss its relevance to colony collapse syndrome. The significance of energetic stress as a general mechanism by which infectious diseases influence host behavior and physiology is discussed.     

Cuticular sclerotization in the honeybee (Apis mellifera adansonii)

Summary A micromethod has been developed for quantitative determinations of ketocatechols released by acid hydrolysis from samples of sclerotized insect cuticle, weighing from 0.1 to 5 mg. The method has been used to follow the changes in yield of ketocatechols from five different types of cuticle during honeybee development. Samples from workers, drones, and queens have been analyzed. The increase in yield of ketocatechols is most pronounced during the period of pharate development when abdominal cuticle is stiffened and the cuticular proteins become insoluble.     

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂ and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.     

Chill-coma and cold death temperatures of Apis mellifera

Worker bees have a lower chill-coma temperature than drones or queens, which is influenced by their acclimatisation temperature as is their food consumption at a given temperature. Most bees die after 50 hrs. in chill coma, and survive longer at 5° C than at 0 or 10° C. Cold death occurs between –2 and –6° C and is unaffected by acclimatisation. Winter bees have lower chill-coma temperatures than summer bees but after acclimatisation to 35° C there is no difference. The chill-coma temperature of summer bees decreases with age irrespective of acclimatisation.

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Zusammenfassung Arbeitsbienen haben eine niedrigere Kältestarretemperatur als Drohnen und Königinnen. Akklimatisierung der Arbeiterinnen an verschiedene Temperaturen beeinflußt ihre Kältestarretemperaturen und die Zeit, die sie zur Erholung von der Kältestarre benötigen. Die Akklimatisation ist nach 24 Stunden vollständig. Die Stoffwechselrate der Bienen bei einer gegebenen Temperatur wird ebenfalls von der Akklimatisationstemperatur beeinflußt.Die meisten Bienen sterben nach 50stündiger Kältestarre und wenige überleben mehr als 80 Stunden. Bei 5° C ist die Überlebensrate größer als bei 0° oder 10°. Der Tod tritt zwischen –2° und –6° C ein und ist von vorangegangener Akklimatisation unbeeinflußt.Bienen vom Rande einer Wintertraube haben niedrigere Kältestarretemperaturen als Bienen aus der Mitte. Winterbienen haben eine niedrigere Kältestarretemperatur als Sommerbienen, doch nach Akklimatisation an 35° C besteht kein Unterschied mehr. Die Kältestarretemperatur nimmt im Sommer mit dem Alter ab, ob die Bienen an 35° C akklimatisiert sind oder nicht. Es wird vermutet, daß dies auf Unterschieden in der Stoffwechselrate der betreffenden Bienen beruht.

     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

The effect of induced changes in sexual asymmetry of honey bees (Apis mellifera) on swarming behaviour

This study was built on the assumption that mother (queen) and workers (nurses) distribute their genes either through swarms (female biomass) or through the drones (male biomass). The swarming mood of the bee colonies was suppressed by an exactly defined increase in drone rearing. We studied the efficiency of reproductive investments (on genetic and energetic levels) of the mother and workers to the next generations. The equalization of fitness of the mother and nurses was achieved by a deliberately induced change in numerically stable sex asymmetry of a bee colony. A swarm was compensated with its energy demand and a volume ratio of distributed genetic information. The newly introduced term “reproductive investment complex” (RIC) includes the reproductive potential of the mother and reproductive energy of workers into care for the mother and for the brood. The number of individuals of one sex was closely connected with the weight of individuals of the oppposite sex. The described method of suppression of swarming mood was successfully tested on 60 honey bee colonies over seven years (2003–2009). A number of beekeepers that were acquainted with this method confirmed the success.     

The effect of the ectoparasitic mite, Varroa destructor on adult worker honeybee (Apis mellifera) emergence weights, water, protein, carbohydrate, and lipid levels

Wet weight, dry weight and water contents of emerging honeybees (Apis mellifera L. [Hymenoptera: Apidae]) infested with the ectoparasitic mite Varroa destructor (Anderson) (Acari: Varroidae) were all negatively correlated with increasing numbers of mites. It was estimated that for every female mite present during the bees' development, the host would lose three percent of its body water. Parasitised bees also emerged with lower head and abdomen concentrations of protein and with lower abdominal carbohydrate concentrations. Lipid concentrations were not detectably affected by V. destructor infestation. The losses of metabolic reserves were not, however, judged to be serious enough to be directly responsible for the high bee mortality and ultimate colony collapse that are associated with the arrival of Varroa in a hive. Some 8.5% of the emerging bees exhibited morphological deformities and deformity was positively correlated with increasing numbers of mites in brood cells. Deformed bees were, however, found in all categories of parasitosis, suggesting that other factors, such as infectious agents, may be involved. Mites that fed on either live or dead U¹⁴C- labelled bees acquired the label within 24 h and it was calculated that an adult female mite consumes 0.67 l haemolymph 24 h^–1. It was also demonstrated that ¹⁴C was transmitted to a previously non-radio-labelled bee when a mite that had been feeding on a labelled bee changed hosts. The level of transfer was above that which could have arisen through contamination of the mites' mouthparts and supports the suggestion that Varroa is an important vector of pathogens such as viruses.     

The honeybee queen influences the regulation of colony drone production

Social insect colonies invest in reproduction and growth, buthow colonies achieve an adaptive allocation to these life-historycharacters remains an open question in social insect biology.Attempts to understand how a colony's investment in reproductionis shaped by the queen and the workers have proved complicatedbecause of the potential for queen–worker conflict overthe colony's investment in males versus females. Honeybees,in which this conflict is expected to be minimal or absent,provide an opportunity to more clearly study how the actionsand interactions of individuals influence the colony's productionand regulation of males (drones). We examined whether honeybeequeens can influence drone regulation by either allowing orpreventing them from laying drone eggs for a period of timeand then examining their subsequent tendency to lay drone andworker eggs. Queens who initially laid drone eggs subsequentlylaid fewer drone eggs than the queens who were initially preventedfrom producing drone eggs. This indicates that a colony's regulationof drones may be achieved not only by the workers, who buildwax cells for drones and feed the larvae, but also by the queen,who can modify her production of drone eggs. In order to betterunderstand how the queen and workers contribute to social insectcolony decisions, future work should attempt to distinguishbetween actions that reflect conflict over sex allocation andthose that reflect cooperation and shared control over the colony'sinvestment in reproduction.     

信息素介导的西方蜜蜂与狄斯瓦螨互作研究进展

狄斯瓦螨是一种严重危害西方蜜蜂的体外寄生螨,是世界养蜂业的最大威胁.人们广泛采用化学方法防治狄斯瓦螨,但易引起狄斯瓦螨的抗药性、蜜蜂中毒和蜂产品药物残留等问题.为此,人们尝试了多种蜂螨绿色防控技术.其中利用蜜蜂信息素防治狄斯瓦螨是一个重要的研究方向.研究表明,狄斯瓦螨能利用蜜蜂信息素识别处于不同发育阶段的寄主,并对特定时期的寄主表现出高度的选择性.近年来,多种能作用于狄斯瓦螨的蜜蜂信息素相继被报道.这些信息素包括成蜂、蛹和幼虫信息素.有的信息素对狄斯瓦螨表现出驱避作用,有的则表现出引诱作用.本文对这些信息素的种类、主要组成成分、对狄斯瓦螨的作用等进行了综述,旨在为今后的研究与应用提供参考.     

蜜蜂对复合气味中特征因素的选择性记忆巩固的研究

目的 蜜蜂天生具有丰富的嗅觉辨识能力,觅食、交配、导航以及社交活动均依赖其嗅觉系统,是研究嗅觉感知和学习记忆的行为及神经机制的理想模型。蜜蜂既能够将某个复合气味作为一个整体也可以将复合气味的各组成成分进行辨别和区分,但是在特征依赖的联合记忆中依据何种原则进行加工并存储到长期记忆还不清楚。方法 本文利用特征阳性（feature positive：AB+,B-）和特征阴性（feature negative：AB-,B+）的奖赏性嗅觉条件化,训练蜜蜂对复合气味和成分气味的辨别,并检测蜜蜂对复合气味（AB）、成分气味（B）以及特征气味（A）的中长时记忆（3 h）和长时记忆（24 h）。结果 在特征阳性的奖赏性嗅觉条件化中,蜜蜂对训练过的气味可以形成稳定的中长时和长时记忆,并且对复合气味中的特征气味的记忆与复合气味的记忆呈现高度相似。但在特征阴性的奖赏性嗅觉条件化中,蜜蜂虽能够在3 h和24 h对训练过的两种气味具有显著的伸喙反应差异,且对特征阴性的气味无显著反应,但对复合气味的反应随时间的推移而增加。结论 实验结果表明,蜜蜂选择性地将与奖赏信息联合出现的气味巩固到长时记忆中,但并未依据特征成分加工储存到长时记忆中。奖赏信息预示着食物源,与生存息息相关,表明对环境信息进行选择性的记忆巩固加工并储存可能是低等动物高效地编码生存相关信息的重要策略。     

The influence of queen age and quality during queen replacement in honeybee colonies

Honeybee, Apis mellifera, colonies replace their queens by constructing many queen cells and then eliminating supernumerary queens until only one remains. The ages of the queens and the variation in their reproductive potential are important factors in the outcome of such events. Selection would favour colonies that requeen as quickly as possible to minimize the brood hiatus, therefore selecting for queens reared from older larvae. Conversely, reproductive potential (queen 'quality') is maximized by rearing queens from younger larvae. This potential trade-off was tested during two phases of queen replacement, namely queen rearing and polygyny reduction. Our results suggest that queen age is a significant element during both queen rearing and polygyny reduction, whereas queen quality, at least to the magnitude tested in this experiment, has little impact on the outcome of either process. The rate of queen replacement therefore appears to be an important factor in the honeybee life cycle, and further mechanisms of potential importance during this life history transition are discussed. Copyright 2000 The Association for the Study of Animal Behaviour.     

东方蜜蜂入侵群体的建立机制

蜜蜂、胡蜂及蚂蚁等是具有高度入侵性的社会性昆虫.理论上,在奠基者群体数量较少时,其单一位点互补性性别决定机制会限制入侵群体的发展.在人类活动影响下,东方蜜蜂已扩散至其自然分布区之外的很多地区,给当地饲养的西方蜜蜂带来很大影响.本文简要介绍了东方蜜蜂的分布、性别决定机制以及近几年来针对澳大利亚东方蜜蜂入侵群体的系列研究结果,从蜂王的交尾行为、工蜂的繁殖行为、平衡选择作用、产雌孤雌生殖等方面分析了东方蜜蜂群体如何在奠基者效应带来的遗传负荷压力下快速发展起来,为揭示相关社会性昆虫入侵群体的建立机制提供了很好的借鉴.     

意大利蜜蜂工蜂幼虫nkd基因的生物信息学分析及功能研究

【目的】意大利蜜蜂(Apis mellifera ligustica,简称意蜂)是西方蜜蜂(Apis mellifera)的亚种之一。蜜蜂球囊菌(Ascosphaera apis)侵染意蜂幼虫导致白垩病。本研究对西方蜜蜂裸表皮蛋白(naked cuticle, Nkd)进行保守基序预测和系统进化分析,并通过RNAi明确nkd基因对意蜂工蜂幼虫体重及宿主响应蜜蜂球囊菌胁迫的免疫应答的影响,以期丰富西方蜜蜂基因nkd的信息,并揭示意蜂幼虫nkd的功能。【方法】通过MEME软件预测西方蜜蜂和其他9个物种Nkd蛋白的保守基序。采用MEGA X软件对西方蜜蜂及其他9个物种的Nkd蛋白进行系统进化分析。通过饲喂dsRNA对意蜂幼虫肠道内的nkd进行RNAi。使用电子天平对幼虫进行称重。利用RT-qPCR检测nkd基因的干扰效率及免疫基因的相对表达量。【结果】西方蜜蜂与东方蜜蜂、柑橘凤蝶、家蚕和金凤蝶的Nkd蛋白均含有3个保守基序(motif 1、motif 2 和motif 3),说明上述5个昆虫物种的Nkd具有较高的保守性。西方蜜蜂与东方蜜蜂的Nkd蛋白聚为一支,说明二者的亲缘关系近。与dsRNA-egfp组相比,dsRNA-nkd组5日龄和6日龄幼虫肠道内nkd的表达量均极显著下调(P<0.001),干扰效率分别为49.60%和56.40%。另外,dsRNA-nkd组幼虫体重较dsRNA-egfp组显著下降,说明nkd显著影响幼虫体重。RT-qPCR结果显示,4日龄幼虫肠道内abaecin、apidaecin、birc5、defensin-1和PGRP-S2均被激活表达;5日龄幼虫肠道内abaecin、apidaecin、birc5和defensin-1均被激活表达,PGRP-S2的表达受到抑制;6日龄幼虫肠道内abaecin被激活表达,而apidaecin、birc5、defensin-1和PGRP-S2的表达均受到抑制,说明上述5个免疫基因在宿主响应胁迫的过程中呈不同的表达趋势,均参与宿主的免疫应答,nkd与abaecin和apidaecin的表达存在负向调控关系。【结论】西方蜜蜂的Nkd蛋白含有3个保守基序(motif 1、motif 2和motif 3),西方蜜蜂与东方蜜蜂的Nkd蛋白亲缘关系最近,通过饲喂dsRNA能有效干扰意蜂工蜂幼虫肠道内nkd表达,nkd影响意蜂工蜂幼虫体重及宿主对蜜蜂球囊菌胁迫的免疫应答。     

中华蜜蜂和意大利蜜蜂感染囊状幼虫病毒后免疫及发育相关基因表达比较

【目的】本文旨在探究囊状幼虫病毒(sacbrood virus, SBV)对中华蜜蜂(Apis cerana cerana,简称中蜂)和意大利蜜蜂(Apis mellifera ligustica,简称意蜂)工蜂幼虫发育和免疫基因、营养代谢基因、抗病毒基因、细胞发育及代谢相关基因表达的影响。【方法】从蜂群中移取2日龄的中蜂和意蜂幼虫,在培养箱(34℃, RH 85%)进行人工饲养,3日龄时接种SBV病毒,每天观察记录死亡情况,并通过实时定量PCR (real-time quantitative polymerase chain reaction, RT-qPCR)检测4日龄和7日龄幼虫体内SBV基因相对表达量,及免疫基因(Apidaecin、Abaecin、Hymenoptaecin、Denfensin、Lys-1、Pgrp-lc、Kenny、Domeless)、营养代谢基因(Ilp1、Hex110、Vg)、抗病毒基因(Dis3、Dicer、Ago1)、细胞组成及发育调控基因(Vhdl、Co-1-iv)以及细胞代谢和调控基因(Mta1)的表达水平。【结果】通过分析比较发现,感染相同剂量...     

西方蜜蜂工蜂肠道菌群定殖起始点和定殖稳态点的结构比较分析

【目的】目前,针对昆虫肠道细菌定殖规律的研究还未见报道。探索西方蜜蜂(Apis mellifera)肠道菌群定殖过程两个重要时间节点(起始0日龄和稳态7日龄)间肠道细菌群落(菌群)结构组成的差异,加深对蜜蜂及昆虫肠道菌群定殖规律的认识。【方法】分别采集两个化蛹后工蜂发育阶段的个体各5只,分别解剖并提取其肠道菌群DNA。使用Illumina技术对肠道菌群16S rD NA高变区进行高通量测序。通过生物信息学的分析方法对肠道菌群进行多样性分析,并对两个时间点相对丰度最高的肠道菌群进行统计分析,比较肠道菌群相对丰度和组成的差异。【结果】共获得515156条高质量序列,长度为227904953bp,平均长度为442bp。基于OTUs的分类表明,工蜂肠道细菌分别隶属于34个门82个纲221个目405个科799个属。此外,工蜂肠道菌群定殖起点和终点间的Alpha多样性指数存在显著差异(ACE,P=0.0014;Chao,P=0.0013;Shannon,P=0.0003;Simpson,P=0.0028,Student’s t检验)。此外,相较0日龄工蜂,7日龄工蜂肠道中的乳酸杆菌Lactobacillus、Gilliamella、双歧杆菌Bifidobacterium、Snodgrassella4个属的相对丰度显著增加;相反,不动杆菌Acinetobacter、大肠杆菌志贺氏菌Escherichia-Shigella、鞘脂单胞菌Sphingomonas、类杆菌Bacteroides、涅斯捷连科氏菌Nesterenkonia、栖热菌Thermus6个属的细菌相对丰度显著降低(P0.05)。【结论】出房(0日龄)成年工蜂的肠道菌群多样性显著高于菌群定殖完成(7日龄)工蜂的肠道菌群多样性,且成年工蜂肠道菌群定殖完成前后部分类群的相对丰度显著改变。本研究的结果不仅可增加我们对蜜蜂肠道菌群定殖规律的认识,也能够为研究其他昆虫肠道菌群的定殖规律提供重要的参考信息。     

Dependence of the life span of the honeybee (Apis mellifica) upon flight performance and energy consumption

Summary The life span of worker-honeybees is determined by the duration of the hive-period and of the foraging period (Figs. 1,2). The duration of the forgaing period is regulated in the following way: Total flight performance of the individual bee seems to be fixed. Daily flight performance strongly affects total flight duration. High daily flight performance decreases maximal flight duration and vice versa.Foragers accumulate the highest glycogen reserves in the flight muscles compared to other stages (Figs. 3, 4). They use these reserves to overcome starvation or when growing old. Young foragers are able to restore glycogen reserves after sugar intake, whereas old foragers were found to have a reduced glycogen synthesizing ability (Fig. 5).The results indicate that bees exhaust their energysupplying mechanisms after a definite total flight performance.