意大利蜜蜂工蜂卵巢发育的研究

蜜蜂Apis mellifera L.蜂群中的工蜂卵巢发育和工蜂产卵现象受多种因素控制,了解其影响因素对养蜂生产具有重要意义。本研究将意大利蜜蜂蜂群设置为囚王群(tg1)、无王有子群(tg2)、无王无子群(tg3)以及正常有王蜂群(CK)4个试验组,通过对工蜂卵巢管的显微观察,确定不同处理组工蜂在不同时间段内卵巢的发育情况。结果表明:随着时间的延长,与CK处理组相比,tg3处理组中的工蜂卵巢发育水平最高,tg2次之,tg1最低;在31d时,4个处理组两两之间差异均达到显著水平(P<0.05)。tg1、tg2和tg3处理组中工蜂产卵前期时间分别为35、22和17d,而CK蜂群在试验期内未出现工蜂产卵现象;tg2和tg3处理组的工蜂产卵的封盖前期时间分别为8和6d,而tg1和CK组在试验期内未出现子房封盖现象。蜂群失王时间过长会刺激工蜂卵巢发育,并导致其产卵;蜂群的短期失王和蜂王老化也会刺激工蜂卵巢发育,但是刺激程度较低;蜂群中的蜂子能抑制工蜂卵巢管的发育,因此在蜂群短时间失王时可以适当地补充子脾延缓工蜂卵巢发育。     

意大利蜜蜂工蜂唾腺和中肠消化酶活性比较研究

对意大利蜜蜂 Apis mellifera ligustica L工蜂唾腺和中肠中淀粉酶、蛋白酶、蔗糖酶、果胶酶和海藻糖酶5种消化酶进行活性测定,发现巾肠的蛋门酶和蔗糖酶活性较唾腺高,有显著性差异;唾腺的果胶酶活性较中肠高,有显著性差异;唾腺与中肠的淀粉酶和海藻糖酶没有显著性差异.表明意大利蜜蜂工蜂对蔗糖和蛋白的消化主要由中肠分泌的消化酶来完成,唾腺起辅助作用;对果胶的消化主要在唾腺,中肠起辅助作用;而在淀粉和海藻糖的消化过程中唾腺和中肠都有很重要的作用.     

意大利工蜂前胃显微结构观察

通过解剖镜和扫描电子显微镜对意大利工蜂Apismellifera ligusticaL.前胃的形态结构进行了观察,发现前胃包括前胃瓣和前胃管两部分。前胃瓣是由4个瓣片环围而成,球形,在蜜囊中,每个瓣片的长度约为0.8mm,前端为三角形结构,可以运动,边长约为0.35mm,三角形的前缘有较长的毛形结构。在每个三角形顶端各有一个直径约为50μm瘤形突起。前胃瓣最大孔径为0.89～1.00mm,具有调整和控制食物进入中肠的功能。前胃管是一直径0.01～0.02mm的较为柔软的管道,插入中肠5mm,该结构可防止中肠食物的倒流。前胃瓣上有7种毛状结构,分布在前胃瓣的不同部位,该结构的功能是分离和过滤、捕捉和感觉功能。本研究为理解工蜂前胃在消化系统中的功能和作用以及昆虫形态学和昆虫分类学提供理论依据。     

蜜蜂无政府主义蜂群的研究进展

西方蜜蜂Apis mellifera作为典型的社会性昆虫, 最重要的特征是生殖劳动分工。蜂王垄断蜂群的生殖权利, 工蜂生殖功能受到抑制, 从事除产卵和交配以外的所有职能。而在无政府主义蜂群中, 即使蜂王存在, 也有较多工蜂的卵巢激活并产卵, 蜂群中大多数雄蜂是工蜂的后代。这些特殊蜂群为正常蜂群工蜂不育机制研究提供了绝佳的反例材料。本文对无政府主义蜂群的行为特征、 产生条件、 遗传基础等研究进行了综述。无政府主义蜂群中有较多的工蜂产卵, 且工蜂所产卵能够逃避工蜂监督, 这种行为的产生受环境、 遗传组成、 基因表达等多种因素的影响, 并且遗传结构体系复杂, 参与调控的基因数量多。无政府主义蜂群行为机制的研究为工蜂不育机制的揭示及其他社会性昆虫工职不育基因的筛选和功能研究提供借鉴。     

意大利蜜蜂不同日龄工蜂脑部三种miRNA的表达水平

【目的】意大利蜜蜂 Apis mellifera ligustica（简称“意蜂”）是社会性昆虫,蜂群中的工蜂表现出年龄依赖性的行为转变,其行为转变的机制一直是研究的热点。本研究分别检测了3种miRNA（ame-let-7, ame-miR-13b和ame-miR-279）在不同日龄意蜂工蜂脑部的表达情况,以期为探究意蜂工蜂年龄依赖性行为变化机制提供重要线索。【方法】通过荧光定量PCR分别检测了不同发育时期（4, 8, 12, 17, 22, 26和30日龄）意蜂工蜂脑部3种miRNA的表达情况,并检验miRNA的表达差异情况。【结果】ame-let-7的表达量随工蜂日龄的增加逐渐降低,在17日龄后其表达量趋于稳定;ame-miR-13b的表达量则随着工蜂日龄的增加逐渐增加,但在26和30日龄的工蜂中稳定表达;ame-miR-279的表达量则呈类似正态分布状态,其中在12日龄工蜂中表达量最高,且在17日龄后稳定表达。这3种miRNA表达均具有明显的时间特异性。【结论】 ame-let-7, ame-miR-13b和ame-miR-279的表达与工蜂年龄依赖性行为变化具有相关性和规律性,对于深入探究miRNA的作用位点具有重要参考价值。     

中华蜜蜂急造王台的工蜂亲属优惠

人为使3群中华蜜蜂(Apis cerana cerana)失王而出现急造王台后,应用3个蜜蜂微卫星位点A14、A107和B124对蜂群、急造王台中的幼虫及其哺育蜂的亚家庭进行鉴别,以此来研究中华蜜蜂急造王台的工蜂亲属优惠.结果显示:在3个实验蜂群Ⅰ、Ⅱ、Ⅲ中分别检测到11、13和14个亚家庭,各蜂群各亚家庭之间的工蜂分布差异不显著.然而各蜂群中急造王台却分别只出现在少数的3、4和5个亚家庭中,各亚家庭之间在王台出现率上存在极显著的差异.另外,各急造王台的所有哺育蜂并非只来自幼虫所在的亚家庭,而是分布在更多的亚家庭里,并且各亚家庭之间差异不显著.以上结果证明:中华蜜蜂急造王台时,在蜂王幼虫的选择过程中存在工蜂亲属优惠行为,但蜂王幼虫与它们的哺育工蜂之间并不存在工蜂亲属优惠     

不同水平亮氨酸对繁殖期意大利蜜蜂蜂群群势及工蜂发育的影响

【目的】评价日粮中不同水平亮氨酸对繁殖期意大利蜜蜂Apis mellifera ligustica蜂群群势及工蜂发育的影响。【方法】选取重庆荣昌本地繁殖期意大利蜜蜂21群,随机分为7组,分别饲喂添加0,15.30,17.50,26.25,35.00,43.75和52.5 g/kg亮氨酸的试验日粮,每12 d测定各组蜂群群势、封盖子量、蜂群取食量和营造王台情况,24 d后测定蜂群采集行为、工蜂初生重、工蜂虫体蛋白质含量及工蜂寿命。【结果】当亮氨酸添加水平为43.75和52.50 g/kg时能够显著提高繁殖期意大利蜜蜂的蜂群群势、封盖子量、蜂群取食量及采集行为(P0.05)。与空白对照组相比,基础日粮组及各亮氨酸添加组的新出房蜂和6日龄幼虫体蛋白质含量较高(P0.05);各亮氨酸添加组的工蜂初生重显著高于空白组(P0.05);不同亮氨酸添加水平对意大利蜜蜂工蜂寿命影响较小。此外,亮氨酸添加水平26.25,35.00和43.75 g/kg能够在饲喂0-24 d时促进蜂群营造自然王台,刺激蜂群分蜂。【结论】配方日粮中43.75和52.5 g/kg的亮氨酸能够显著促进繁殖期意大利蜜蜂蜂群发展,其中43.75 g/kg的亮氨酸饲喂量还能刺激蜂群营造自然王台,激发蜂群分蜂。     

工蜂与蜂王的“繁殖等级之战”

蜂群通常具有很高水平的合作,而最新发表在《分子生态学》上的研究表明工蜂和蜂王之间存在繁殖冲突,一些工蜂会“自私”地将蜂群的资源占为己有。该研究以巴西的一种高度社会化的无刺蜂Melipona scutellaris为研究对象,其蜂群有大约1500只工蜂和1只蜂王。     

中华蜜蜂工蜂视叶的胚后发育

为了研究蜜蜂视叶的胚后发育模式, 本研究通过形态解剖、免疫组织化学技术, 对中华蜜蜂Apis cerana cerana工蜂视叶的胚后发育过程进行了系统的比较研究。结果表明: 中华蜜蜂的视叶起源自幼虫早期脑内部的两个视原基。 外部视原基经过不对称的细胞分裂产生神经节母细胞, 随后这些细胞经过快速的对称分裂, 复制自身并生成视髓层神经细胞; 外部视原基的极少数细胞分裂产生视神经节层神经节母细胞, 到蛹发育中期, 随着视神经进入的刺激, 神经节层神经细胞才开始快速增殖, 并最终形成了视神经节层的所有结构。 内部视原基的分裂方式同外部视原基相同, 最终生成视叶的视小叶部分。本研究结果提示中华蜜蜂的视叶起源自两个视原基, 大多数神经细胞在前蛹期产生, 视神经的进入刺激了视神经节层的发育。     

意大利蜜蜂工蜂中肠piRNA的鉴定与分析

【目的】Piwi蛋白互作RNA(PIWI-interacting RNA, piRNA)在昆虫的发育和免疫等重要生物学过程发挥重要的调控作用。本研究旨在丰富西方蜜蜂Apis mellifera的piRNA信息,并为进一步探究差异表达piRNA(differentially expressed piRNA, DEpiRNA)调控意大利蜜蜂Apis mellifera ligustica工蜂中肠发育的分子机理提供基础。【方法】基于已获得的意大利蜜蜂7日龄(Am7)和10日龄(Am10)工蜂中肠的small RNA(sRNA)组学数据,对piRNA进行预测和分析。将质控后的sRNA数据比对西方蜜蜂参考基因组,再将比对上的序列标签(tags)进一步比对数据库以滤除rRNA和tRNA等小非编码RNA(non-coding RNA,ncRNA),进而根据piRNA的长度特征鉴定piRNA。采用TPM(tags per million)算法对piRNA表达量进行计算和归一化处理。根据|log₂ fold change|≥1且P≤0.05的标准筛选Am7 vs Am10 比较组的DEpiRNA。通过相关软件预测DEpiRNA的靶mRNA并进行GO和KEGG数据库注释。利用Cytoscape软件对DEpiRNA-mRNA调控网络进行可视化。通过Stem-loop RT-PCR对随机选取的Am7与Am10两组共有的6个piRNA的表达进行验证。利用RT-qPCR对随机挑选的6个DEpiRNA的表达趋势进行验证。【结果】从意大利蜜蜂工蜂中肠中共鉴定到596个piRNA,长度介于24~33 nt,且Am7和Am10组中不同长度分布范围的piRNA的首位碱基偏向性具有明显差异。在Am7 vs Am10比较组共筛选出41个DEpiRNA,其中piR-ame-11093, piR-ame-1111451,piR-ame-190949和piR-ame-932156可分别靶向1 195, 1 018, 4 040和1 063条mRNA。上述靶mRNA可分别注释到45个功能条目和45条通路。Stem-loop RT-PCR验证结果显示6个piRNA(piR-ame-1084826, piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500和piR-ame-774987)均真实表达。RT-qPCR结果显示共有6个DEpiRNA (piR-ame-1084826,piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500和piR-ame-774987)的表达趋势与测序数据中的表达趋势一致,证实了本研究中sRNA-seq数据的可靠性和piRNA差异表达趋势的真实性。【结论】本研究从意大利蜜蜂工蜂中肠中鉴定到596个piRN,长度介于24~33 nt。不同长度分布范围的piRNA的首位碱基偏向性具有明显差异;piR-ame-11093, piR-ame-1111451,piR-ame-190949和piR-ame-932156具有靶向调控基因表达参与工蜂中肠发育过程的潜力。     

A test of worker policing theory in an advanced eusocial wasp, Vespula rufa

Mutual policing is an important mechanism for maintaining social harmony in group-living organisms. In some ants, bees, and wasps, workers police male eggs laid by other workers in order to maintain the reproductive primacy of the queen. Kin selection theory predicts that multiple mating by the queen is one factor that can selectively favor worker policing. This is because when the queen is mated to multiple males, workers are more closely related to queen's sons than to the sons of other workers. Here we provide an additional test of worker policing theory in Vespinae wasps. We show that the yellowjacket Vespula rufa is characterized by low mating frequency, and that a significant percentage of the males are workers' sons. This supports theoretical predictions for paternities below 2, and contrasts with other Vespula species, in which paternities are higher and few or no adult males are worker produced, probably due to worker policing, which has been shown in one species, Vespula vulgaris. Behavioral observations support the hypothesis that V. rufa has much reduced worker policing compared to other Vespula. In addition, a significant proportion of worker-laid eggs were policed by the queen.     

Worker policing in the German wasp Vespula germanica

In some ants, bees, and wasps, workers kill or "police" maleeggs laid by other workers in order to maintain the reproductiveprimacy of the queen. Kin selection theory predicts that multiplemating by the queen is one factor that can selectively favorworker policing. This is because when the queen is mated tomultiple males, workers are more closely related to the queen'ssons than to the sons of other workers. Earlier work has suggestedthat reproductive patterns in the German wasp Vespula germanicamay contradict this theory, because in some colonies a largefraction of the adult males were inferred to be the workers'sons, despite the effective queen mating frequency being greaterthan 2 (2.4). In the present study, we reexamine the V. germanicacase and show that it does support the theory. First, geneticanalysis confirms that the effective queen mating frequencyis high, 2.9, resulting in workers being more related to thequeen's sons than to other workers' sons. Second, behavioralassays show that worker-laid eggs are effectively killed byother workers, despite worker-laid eggs having the same intrinsicviability as queen-laid ones. Finally, we estimate that approximately58.4% of the male eggs but only 0.44% of the adult males areworker derived in queenright colonies, consistent with workerreproduction being effectively policed.     

Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming

Kin selection theory predicts that honeybee (Apis mellifera) workers should largely refrain from producing their own offspring, as the workers collectively have higher inclusive fitness if they rear the sons of their mother, the queen. Studies that have quantified levels of ovary activation and reproduction among workers have largely supported this prediction. We sampled pre‐emergent male pupae and adult workers from seven colonies at regular intervals throughout the reproductive part of the season. We show that the overall contribution of workers to male (drone) production is 4.2%, nearly 40 times higher than is generally reported, and is highest during reproductive swarming, when an average of 6.2% of the males genotyped are worker‐produced. Similarly, workers in our samples were 100 times more likely to have active ovaries than previously assumed. Worker reproduction is seasonally influenced and peaks when colonies are rearing new queens. Not all worker subfamilies contribute equally to reproduction. Instead, certain subfamilies are massively over‐represented in drone brood. By laying eggs within the period in which many colonies produce virgin queens, these rare worker subfamilies increase their direct fitness via their well‐timed sons.     

Colony stage and not facultative policing explains pattern of worker reproduction in the Saxon wasp

Inclusive fitness theory predicts that in colonies of social Hymenoptera headed by a multiple‐mated queen, workers should benefit from policing eggs laid by other workers. Foster & Ratnieks provided evidence that in the vespine wasp Dolichovespula saxonica, workers police other workers’ eggs only in colonies headed by a multiple‐mated queen, but not in those headed by a single‐mated one. This conclusion, however, was based on a relatively small sample size, and the original study did not control for possible confounding variables such as the seasonal colony progression of the nests. Our aim, therefore, was to reinvestigate whether or not facultative worker policing occurs in D. saxonica. Remarkably, our data show that in the studied Danish population, there was no correlation between worker–worker relatedness and the percentage of worker‐derived males. In addition, we show that variability in cuticular hydrocarbon profiles among the workers did not significantly correlate with relatedness and that workers therefore probably did not have sufficient information on queen mating frequency from the workers’ cuticular hydrocarbon profiles. Hence, there was no evidence that workers facultatively policed other workers’ eggs in response to queen mating frequency. Nevertheless, our data do show that the seasonal progression of the nest and the location in which the males were reared both explain the patterns of worker reproduction found. Overall, our results suggest that the earlier evidence for facultative worker policing in D. saxonica may have been caused by accidental correlations with certain confounding variables, or, alternatively, that there are large interpopulation differences in the expression of worker policing.     

Anarchy in the UK: Detailed genetic analysis of worker reproduction in a naturally occurring British anarchistic honeybee,Apis mellifera,colony using DNA microsatellites

Anarchistic behaviour is a very rare phenotype of honeybee colonies. In an anarchistic colony, many workers' sons are reared in the presence of the queen. Anarchy has previously been described in only two Australian colonies. Here we report on a first detailed genetic analysis of a British anarchistic colony. Male pupae were present in great abundance above the queen excluder, which was clearly indicative of extensive worker reproduction and is the hallmark of anarchy. Seventeen microsatellite loci were used to analyse these male pupae, allowing us to address whether all the males were indeed workers' sons, and how many worker patrilines and individual workers produced them. In the sample, 95 of 96 of the males were definitely workers' sons. Given that approximately 1% of workers' sons were genetically indistinguishable from queen's sons, this suggests that workers do not move any queen-laid eggs between the part of the colony where the queen is present to the area above the queen excluder which the queen cannot enter. The colony had 16 patrilines, with an effective number of patrilines of 9.85. The 75 males that could be assigned with certainty to a patriline came from 7 patrilines, with an effective number of 4.21. They were the offspring of at least 19 workers. This is in contrast to the two previously studied Australian naturally occurring anarchist colonies, in which most of the workers' sons were offspring of one patriline. The high number of patrilines producing males leads to a low mean relatedness between laying workers and males of the colony. We discuss the importance of studying such colonies in the understanding of worker policing and its evolution.     

Egg marking pheromones of anarchistic worker honeybees (Apis mellifera)

In honeybees, worker policing via egg eating enforces functionalworker sterility in colonies with a queen and brood. It is thoughtthat queens mark their eggs with a chemical signal, indicatingthat their eggs are queen-laid. Worker-laid eggs lack this signaland are, therefore, eaten by policing workers. Anarchistic workerhoneybees have been hypothesized to circumvent worker policingby mimicking the queen egg-marking signal. We investigated thisphenomenon by relating chemical profiles of workers and theireggs to egg acceptability. We found that the ability of someworkers (anarchistic workers in queenright colonies and deviantworkers from a queenless colony) to lay more acceptable eggsis due to them producing significant amounts of queen-like estersfrom their Dufour's gland. These esters appear to be transferredto eggs during laying and increase egg survival. However, theseesters cannot be the normal queen egg-marking signal, as theyare generally absent from queen-laid eggs and only increasethe short-term persistence of worker-laid eggs, because only7–30% of anarchistic worker-laid eggs persisted to hatchingversus 91–92% of queen-laid eggs. All workers can producesome esters, but only workers that greatly increase their esterproduction lay more acceptable eggs. The production of estersappears to be a flexible response, as anarchistic workers rearedin queenless colonies did not increase their ester production,while some deviant workers in queenless colonies did increasetheir ester production.     

A quantitative study of worker reproduction in queenright colonies of the Cape honey bee, Apis mellifera capensis

Reproduction by workers is rare in honey bee colonies that have an active queen. By not producing their own offspring and preventing other workers from producing theirs, workers are thought to increase their inclusive fitness due to their higher average relatedness towards queen-produced male offspring compared with worker-produced male offspring. But there is one exception. Workers of the Cape honey bee, Apis mellifera capensis, are able to produce diploid female offspring via thelytokous parthenogenesis and thus produce clones of themselves. As a result, worker reproduction and tolerance towards worker-produced offspring is expected to be more permissive than in arrhenotokous (sub)species where worker offspring are male. Here we quantify the extent to which A. m. capensis workers contribute to reproduction in queenright colonies using microsatellite analyses of pre-emergent brood. We show that workers produced 10.5% of workers and 0.48% of drones. Most of the workers' contribution towards the production of new workers coincided with the colonies producing new queens during reproductive swarming.     

Cape honeybees, Apis mellifera capensis, police worker-laid eggs despite the absence of relatedness benefits

In the Cape honeybee, Apis mellifera capensis, workers lay diploid(female) eggs via thelytoky. In other A. mellifera subspecies,workers lay haploid (male) eggs via arrhenotoky. When thelytokousworker reproduction occurs, worker policing has no relatednessbenefit because workers are equally related to their sisterworkers' clonal offspring and their mother queen's female offspring.We studied worker policing in A. m. capensis and in the arrhenotokousAfrican honeybee A. m. scutellata by quantifying the removalrates of worker-laid and queen-laid eggs. Discriminator coloniesof both subspecies policed worker-laid eggs of both their ownand the other subspecies. The occurrence of worker policing,despite the lack of relatedness benefit, in A. m. capensis stronglysuggests that worker reproduction is costly to the colony andthat policing is maintained because it enhances colony efficiency.In addition, because both subspecies policed each others eggs,it is probable that the mechanism used in thelytokous A. m.capensis to discriminate between queen-laid and worker-laideggs is the same as in arrhenotokous A. m. scutellata.     

Worker reproduction and policing in insect societies: an ESS analysis

Insect societies are vulnerable to exploitation by workers who reproduce selfishly rather than help to rear the queen's offspring. In most species, however, only a small proportion of the workers reproduce. Here, we develop an evolutionarily stable strategy (ESS) model to investigate factors that could explain these observed low levels of reproductive exploitation. Two key factors are identified: relatedness and policing. Relatedness affects the ESS proportion of reproductive workers because laying workers generally work less, leading to greater inclusive fitness costs when within-colony relatedness is higher. The second key factor is policing. In many species, worker-laid eggs are selectively removed or 'policed' by other workers or the queen. We show that policing not only prevents the rearing of worker-laid eggs but can also make it unprofitable for workers to lay eggs in the first place. This can explain why almost no workers reproduce in species with efficient policing, such as honeybees, Apis, and the common wasp, Vespula vulgaris, despite relatively low relatedness caused by multiple mating of the mother queen. Although our analyses focus on social insects, the conclusion that both relatedness and policing can reduce the incentive for cheating applies to other biological systems as well.