Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

Conservation of nutrients in larval tissue by cannibalizing honey bees

ABSTRACT. ¹⁴C-phenylalanine appeared in royal jelly secreted by worker honey bees ( Apis mellifera , L.) directly after eating ¹⁴-phenyl-alanine labelled larvae. The amount of labelled phenylalanine in royal jelly was associated directly with the volume of jelly produced. Loss of radio-label from the workers, in the form of O₂¹⁴C, increased when royal jelly secretion ended. These patterns show that conservation of nutrients in cannibalized tissue is enhanced when the cannibal bees are producing royal jelly. Specific activity of radiolabel in worker bee hypopharyngeal glands was higher than in thoracic muscle, showing selective movement within the bees of a nutrient acquired by cannibalism.     

Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4^th to 6^th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.     

王浆主蛋白MRJP7基因在意大利蜜蜂体内的表达

王浆蛋白是蜂王浆生物功能的物质基础,是由王浆蛋白基因家族(mrjps)编码合成的。但部分家族成员如MRJP7在王浆中的含量极少甚至检测不到。基因功能与其在生物体内的时空表达特性相关,为探究mrjp7的生物学功能,本研究利用荧光定量PCR技术对mrjp7在不同发育时期的工蜂和成年工蜂、雄蜂和蜂王的不同组织部位的表达进行定量检测。结果显示mrjp7在成年雄蜂体内的表达水平最低,成年蜂王次之,且在它们的各不同组织部位之间的表达量差异较小。该基因在工蜂幼虫和蛹期的表达同样较低,但在羽化后9日龄前后的哺育蜂王浆腺和头部特异性高表达,这与哺育蜂分泌蜂王浆哺育幼虫和蜂王的功能是相适应的,该结果在转录水平上证实了mrjp7的营养功能,为进一步的研究和应用打下了理论基础。     

西方蜜蜂不同级型王浆主蛋白MRJP8基因的表达差异

王浆主蛋白在蜜蜂的级型分化中具有重要的功能。为探究mrjp8在西方蜜蜂Apis mellifera不同级型的表达模式及功能差异。【方法】 利用荧光定量PCR技术对西方蜜蜂工蜂、 雄蜂和蜂王不同发育时期和不同组织的mrjp8表达水平进行检测。【结果】 工蜂体内mrjp8在9日龄前后的毒腺组织内特异性高表达, 为参照基因表达量的上万倍, 在其他发育时期和组织的表达量则明显较低, 其表达具有明显的时空特异性; 在雄蜂体内其表达量与对照相当; 在蜂王体内表达量可达参照的近1 000倍, 没有组织特异性。【结论】 mrjp8的这种表达模式提示其在工蜂防御及维系蜂王长寿命方面有积极作用, 这为进一步研究该基因乃至整个王浆蛋白基因家族的进化和功能分化提供了依据。     

Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera

Synthetic juvenile hormone (methyl trans,trans,cis-10-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, 1 μg/μl acetone per animal) (JH) was topically applied to 2- to 3-day-old worker honeybee larvae in the hive. Eighty per cent of the hormone-treated larvae were removed from their brood cell before pupation. Only 1 out of 42 adults showed characteristics of an intercaste. Fifty per cent of the control larvae (1 μl acetone) developed to adults, all of which were workers.After topical application of JH and feeding on royal jelly under in vitro conditions, the rate of survival is high (up to 85 per cent adults), but up to 67 per cent of queens and 44 per cent of workers exhibit eye malformations with characteristics of somatic mutation. Formation of a more solid web by the spinning larvae, shortening of the diapause by 1 to 2 days, and unusual shapes of mandibles, legs, and abdomen are a consequence of hormone treatment. The effects are less marked after application of 0·1 instead of 1 μg hormone or after its addition to the food (2 μg/g royal jelly). Treatment of the 2- to 3-day-old worker larvae and subsequent rearing on royal jelly is followed by a shift in caste differentiation from queens and workers to intercastes. In no case, are more queens developed after juvenile hormone treatment.Queen bee determinator, partially purified from royal jelly, induces a concentration-dependent shift from workers to queen differentiation. A threefold increase in the natural determinator concentration of royal jelly results in an almost exclusive (98 per cent) queen formation from 2- to 3-day-old worker larvae. In contrast to this direct effect, the influence of JH is explained as an indirect morphogenetic effect not directly coupled with honeybee caste differentiation.     

Epigenetic and endocrine determinants of lifespan differences between the castes of social insects

Social insects are promising model organisms in the study of longevity mechanisms. They have a caste system, in which the same genomes may produce phenotypes significantly distinguished by longevity. The ontogenetic trajectories of bees depend on the duration of supply by a specific nutrient mixture (royal jelly) at the larval stage. Longer feeding by the royal jelly leads to the formation of the queen epigenome, which is different from the epigenome of a working bee. Such epigenetic differences, in turn, induce endocrine changes manifested in increased synthesis of juvenile hormone and activation of TOR (target of rapamycin) signaling pathway, as well as in the modulation of insulin/IGF-1 pathway in queen-destined larvae. In adults, these processes influence the synthesis of vitellogenin (egg yolk precursor determining many aspects of insect ontogenesis). Epigenetic and andocrine mechanisms that underlie differences in longevity among the castes of social insects are discussed.     

蜂王幼虫与工蜂幼虫发育期食物消耗量的研究

以意大利蜜蜂Apis mellifera ligustica雌性幼虫为试验材料,用鲜王浆、葡萄糖、果糖和酵母抽提物分别配制不同日龄的蜂王幼虫食物和工蜂幼虫食物,采用室内人工饲养蜜蜂幼虫技术饲养12h以内的蜂王幼虫和工蜂幼虫,直到幼虫出现排便和吐丝行为,记录幼虫生长发育期间消耗的总食物量。结果表明:1只蜂王幼虫在生长发育期平均消耗196.8mg蜂王幼虫食物,而1只工蜂幼虫在生长发育期平均消耗139.4mg工蜂幼虫食物。     

Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family

In the honeybee, Apis mellifera, the queen larvae are fed with a diet exclusively composed of royal jelly (RJ), a secretion of the hypopharyngeal gland of young worker bees that nurse the brood. Up to 15% of RJ is composed of proteins, the nine most abundant of which have been termed major royal jelly proteins (MRJPs). Although it is widely accepted that RJ somehow determines the fate of a female larva and in spite of considerable research efforts, there are surprisingly few studies that address the biochemical characterisation and functions of these MRJPs. Here we review the research on MRJPs not only in honeybees but in hymenopteran insects in general and provide metadata analyses on genome organisation of mrjp genes, corroborating previous reports that MRJPs have important functions for insect development and not just a nutritional value for developing honeybee larvae.     

Sterols of organs involved in brood food production and of royal jelly in honey bees

The sterols of the organs (hypopharyngeal and mandibular glands and honey stomachs) involved in worker and queen honey bee brood food production, royal jelly and intact nurse bees were analyzed to obtain information on the selective transfer of specific sterols from one generation to the next. No appreciable increase in the percentage of 24-methylenecholesterol, relative to the total sterols isolated from intact honey bee (Apis mellifera L.), prepupae or adults, was found in the hypopharyngeal or mandibular glands or the honey stomachs from nurse bees reared in colonies fed a chemically-defined diet supplemented with 24-methylenecholesterol. The sterols of these organs contained higher levels of cholesterol than did the sterols of whole body extracts. The other major sterols, sitosterol and isofucosterol, occurred at relative concentrations comparable to whole body extracts. Also, there were higher levels of cholesterol in the sterols from glandular tissues of nurse bees maintained on pollen and sucrose solution than in sterols isolated from intact insects. In a separate study, royal jelly collected over a 6-day period had much higher relative percentages of 24-methylenecholesterol and lower levels of sitosterol and isofucosterol than did the pollen fed to these colonies. The sterols of nurse bees in the latter study had an intermediate concentration of 24-methylenecholesterol. The significance of these findings relative to the unique selective transfer of specific sterols from the diet or from endogenous sterol pools of the nurse bees from generation to generation in the honey bee is discussed.     

The role of exogenous JH I,JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae

Worker larvae at an age of 4½ days were fed one of several mixtures of reconstituted royal jelly adjusted to a refractive index of 1.3825 and supplemented with JH I, JH III or Anti-JH (precocene II). In addition, juvenile hormone was topically applied to larvae of the same age. It was readily apparent that caste induction is concentration-dependent and that 4?-day-old worker larvae can still develop into queens under laboratory conditions, providing that they have not stopped feeding or can be induced to commence feeding again. These findings are contrary to the general belief that queen induction is not possible after a socalled sensitive period of 3–3½ days. Queens resulted only from honey bee larvae exposed to royal jelly containing 1 μg of JH I. In addition, oral application at this concentration resulted in the only case in which the normal mean weights of worker honey bees were exceeded. All other concentrations of juvenile hormone were not sufficient to initiate queen induction, although its lower concentration may have influenced the production of intercastes.Precocene II did not play a role in queen induction and it also did not interfere with the growth of developing larvae or adults. In addition, the lack of malformations in honey bees treated with precocene II indicates that the use of such a compound as a control agent in insect populations will probably not be detrimental to honey bee larvae that are at least 4½ days old. However, large doses of precocene will quickly kill most 3½-day-old honey bee larvae.The evidence presented here clearly indicates that caste determination is regulated by the endocrine system in honey bee larvae. Food intake in honey bee larvae may well be regulated by the endocrine system. Thus, an apparently inhibited corpus allatum (C.A.) could be reactivated by food intake coupled with juvenile hormone. The food intake restriction that worker larvae normally encounter in the hive probably results in a cessation of C.A. activity. The increase in food intake by queen larvae, on the other hand, carries an increase in growth and accompanying morphological changes necessary for queen development. This concept may also explain the development of intercastes encountered in in vitro studies. Only those larvae that follow a normal food intake sequence, i.e. moderate during the first 3–4 days or so, will develop into queens. Conversely, those larvae that take in too much food during the early portion of development may achieve incomplete development of the neurosecretory system and, thus, develop into intercastes.     

感染囊幼病的工蜂咽下腺细胞的超微结构的变化

前言 中华蜜蜂囊幼病是我国养蜂业的一种重要病毒病,发病率很高,有的地区造成大量的幼虫死亡,给我国养蜂生产带来一定的危害,为了对防治病害提供科学的依据,我们首先对该病病原进行了分离、提纯及电子显微镜的研究等工作。发现该病毒对幼虫和成蜂,尤其对工蜂体内各个器官都有程度不同的影响。在幼虫发病期症状特别明显,病幼虫身体松软多水,其表皮容易破裂,当悬挂幼虫时其幼虫末端积聚有透明的液滴,在巢房内幼虫头部尖并变成黑色,头部稍微向上抬起呈船形。死后的幼虫干涸变为褐色的外壳留于巢房内,当感染的幼虫全部封盖后,巢房盖的中央有一小孔,这些异常的变化都是囊幼病的典型症状。幼虫症状虽很明显,但工蜂感染病毒后在外部形态上却没有明显     

Behavioural,physiological and molecular changes in alloparental caregivers may be responsible for selection response for female reproductive investment in honey bees

Reproductive investment is a central life history variable that influences all aspects of life. Hormones coordinate reproduction in multicellular organisms, but the mechanisms controlling the collective reproductive investment of social insects are largely unexplored. One important aspect of honey bee (Apis mellifera) reproductive investment consists of raising female‐destined larvae into new queens by alloparental care of nurse bees in form of royal jelly provisioning. Artificial selection for commercial royal jelly production over 40 years has increased this reproductive investment by an order of magnitude. In a cross‐fostering experiment, we establish that this shift in social phenotype is caused by nurse bees. We find no evidence for changes in larval signalling. Instead, the antennae of the nurse bees of the selected stock are more responsive to brood pheromones than control bees. Correspondingly, the selected royal jelly bee nurses are more attracted to brood pheromones than unselected control nurses. Comparative proteomics of the antennae from the selected and unselected stocks indicate putative molecular mechanisms, primarily changes in chemosensation and energy metabolism. We report expression differences of several candidate genes that correlate with the differences in reproductive investment. The functional relevance of these genes is supported by demonstrating that the corresponding proteins can competitively bind one previously described and one newly discovered brood pheromone. Thus, we suggest several chemosensory genes, most prominently OBP16 and CSP4, as candidate mechanisms controlling queen rearing, a key reproductive investment, in honey bees. These findings reveal novel aspects of pheromonal communication in honey bees and explain how sensory changes affect communication and lead to a drastic shift in colony‐level resource allocation to sexual reproduction. Thus, pheromonal and hormonal communication may play similar roles for reproductive investment in superorganisms and multicellular organisms, respectively.     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

The chemical basis of honeybee, Apis mellifera, caste formation. Partial purification of queen bee determinator from royal jelly

On royal jelly, 1- to 2-day-old honeybee worker larvae have been reared in vitro to adults in a yield of 67±18 per cent. Up to 100 per cent and, on an average, 60 per cent of them were queens and intercaster. The preparation of a basic food from royal jelly by extensive alcohol extraction is described. With this control food, a survival rate of 47±18 per cent was achieved; 15 per cent of the adults were determined, 4·3 per cent of them were queens. Rearing of 1- to 2-day-old worker larvae on a basic food, to which unknown fractions may be added, was used as a biological test for the partial purification of queen bee determinator from royal jelly. By chromatography of the ethanol extract, previously treated with charcoal, on the cation exchanger Dowex 50 WX4 and rechromatography on silica gel, a 10⁵-fold purification of determinator was achieved. Chemical properties of the highly hydrophilic, low molecular active fraction are described.     

Methionine as a methyl donor regulates caste differentiation in the European honey bee (Apis mellifera)

Nutrition contributes to honey bee caste differentiation, but the role of individual nutrients is still unclear. Most essential amino acid contents, except that of methionine (Met), are greater in royal jelly than worker jelly. After ∼3.5 d, the Met content in the latter was slightly greater than in the former. Met is the major raw material used in the synthesis of S-adenosyl-L-methionine, an active methyl donor for DNA methylation, which is an epigenetic driver of caste differentiation. Here, we tested whether Met regulates caste differentiation in honey bees by determining its effects on the caste development of bees receiving four diets: the basic, basic + 0.2% Met, basic + 0.2% Met + 20 mg/kg 5-azacytidine, and basic + 20 mg/kg 5-azacytidine. The presence of Met decreased the adult bee body length and the numbers of ovarioles, indicating that Met may direct the development of female larvae toward worker bees. The upregulated expression of SAMS, Dnmt1, and Dnmt3 caused by Met exposure in 4-d-old larvae indicated that the worker-inductive effects of Met may occur through the promotion of DNA methylation. We investigated the co-effects of Met and glucose on bee development, and found that the effects of an increased glucose level on the number of ovarioles and body length did not strengthen the worker-inductive effects caused by Met. Our results contribute to caste development theory and suggest that Met—as a methyl donor—plays a regulatory, but not decisive, role in caste differentiation.     

Laying workers in queenless honeybee (Apis mellifera L.) colonies have physiological states similar to that of nurse bees but opposite that of foragers

Honeybee workers shift their labors from nursing their brood to foraging according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiological state of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and hemolymph vitellogenin titers. In a normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high hemolymph vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 70-kDa alpha-glucosidase and low hemolymph vitellogenin titers. In queenless colonies, however, workers with developed ovaries (laying workers) tended to have more developed HPGs and to synthesize major royal jelly proteins, whereas workers with shrunken HPGs tended to synthesize alpha-glucosidase and to have undeveloped ovaries. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers. These findings indicate that the physiological state of laying workers is similar to that of nurse bees, but opposite that of foragers.     

Diet and cell size both affect queen-worker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae)

Background

Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation.

Methodology/Principal Findings

We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62.

Conclusions/Significance

We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same.     

蜜蜂级型分化机理

蜜蜂Apis spp.能有效地为多种植物及农作物授粉, 具有重要的经济和生态价值; 蜜蜂作为高度真社会性昆虫, 已成为社会生物学研究的模式生物。社会性昆虫的生殖劳动分工具有重要的进化意义, 而级型分化是形成生殖劳动分工的基础。近年来, 关于蜜蜂级型分化的研究已取得诸多重要成果, 其机理也得到了较为深入的阐释。营养差异引发蜜蜂幼虫的级型分化。蜂王浆中的主要蛋白组分之一--Royalactin是诱导蜂王发育的关键营养因子, 而脂肪体细胞的表皮生长因子受体介导了Royalactin的这种蜂王诱导作用。DNA甲基化是重要的表观遗传机制之一, 且与个体发育和疾病发生紧密相关, 近来的研究表明DNA甲基化在蜜蜂级型分化过程中发挥重要的调控作用。此外, 越来越多的研究进一步深化了人们对内分泌系统调节级型分化作用的认识。本文从关键营养因子调控、 表观遗传调控和内分泌调节3方面综述蜜蜂级型分化的机理, 并对未来的研究提出可能的方向。     

Effect of royal jelly on longevity and memory-related traits of Apis mellifera workers

Royal jelly (RJ) is a key factor for honey bee caste determination. The queen bee is fed with RJ by worker bees throughout her life, while the worker bees eat bee bread themselves. This study was designed to explore the effect of nutrient-rich RJ on longevity and learning and memory abilities of workers of the western honey bee Apis mellifera. The newly emerged worker bees were randomly divided into three groups and were fed 50% sucrose solution containing 0%, 10%, and 20% RJ. We found that worker bees fed with 10% and 20% RJ showed significantly improved longevity and higher proboscis extension response success rate compared to bees fed with 50% sucrose containing 0% RJ. Additionally, bees fed with 20% RJ showed significantly higher level of expression of memory related genes (GluRA and Nmdar1) compared to the control group. Furthermore, expression of the Nmdar1 gene of worker bees fed with 10% RJ was also significantly higher than in the control group. These results indicate that RJ has potential effects on the longevity and learning and memory abilities of A. mellifera.