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.     

饲喂胡萝卜和蜂王浆对黄粉虫繁殖力的影响

采用多因素对比饲养试验,设计不同黄粉虫TemibriomolitorL.发育时期分别补饲胡萝卜、不同浓度的蜂王浆,研究胡萝卜和蜂王浆对黄粉虫繁殖力的影响。结果表明:各处理组比对照组羽化齐一,羽化率增高,产卵速度加快,总产卵量增多,良卵率、孵化率增高。幼虫期和成虫期都补饲2%的蜂王浆,可使产卵量极显著高于对照97.79%、孵化率极显著地高于对照8.90%;而只在成虫期补充2%的蜂王浆,也可使产卵量极显著高于对照90.65%、有效地提高孵化率6.66%。     

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.     

Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees

Worker and queen bees are genetically indistinguishable. However, queen bees are fertile, larger and have a longer lifespan than their female worker counterparts. Differential feeding of larvae with royal jelly controls this caste switching. There is emerging evidence that the queen-bee phenotype is driven by epigenetic mechanisms. In this study, we show that royal jelly--the secretion produced by the hypopharyngeal and mandibular glands of worker bees--has histone deacetylase inhibitor (HDACi) activity. A fatty acid, (E)-10-hydroxy-2-decenoic acid (10HDA), which accounts for up to 5% of royal jelly, harbours this HDACi activity. Furthermore, 10HDA can reactivate the expression of epigenetically silenced genes in mammalian cells. Thus, the epigenetic regulation of queen-bee development is probably driven, in part, by HDACi activity in royal jelly.     

蜂王浆主蛋白研究进展

作为社会性昆虫,蜜蜂是研究社会行为和学习记忆的理想模式生物。王浆主蛋白（Major royal jelly protein, MRJP）是蜂王浆中蛋白质的主要成分,该家族一共有9个成员,MRJP1~MRJP9。所有mrjps均以串联排列的形式位于蜜蜂11号染色体上一个大约60 kb的DNA片段上。mrjp的同源体也存在于其他的膜翅目昆虫,均是通过yellow进化而来的。随着不断地进化,MRJPs家族进化出许多重要功能,其中最主要的就是营养功能。本文从MRJPs家族的基因及蛋白质结构、mRNA表达情况、进化和功能等方面进行综述,为今后开展相关研究提供理论支持。     

Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by Indirect Enzyme-Linked Immunosorbent Assay

Rabbit polyclonal antibody produced by a major royal jelly protein 1 (MRJP1) specific peptide reacted only with a MRJP1. Indirect ELISA with the antibody revealed a MRJP1 level of 4.12–4.67 g/100 g in different company's royal jelly, which almost agreed with that of a hexametric form of MRJP1 (apisin) measured by high performance liquid chromatography. These results suggest that MRJP1 exists mainly as apisin in royal jelly.     

Trace and mineral elements in royal jelly and homeostatic effects

Royal jelly from Apis mellifera is a highly active natural biological substance and is probably one of the most interesting raw substances in natural product chemistry. Trace elements play a key role in the biomedical activities associated with royal jelly, as these elements have a multitude of known and unknown biological functions. For this reason concentrations of 28 trace (Al, Ba, Sr, Bi, Cd, Hg, Pb, Sn, Te, Tl, W, Sb, Cr, Ni, Ti, V, Co, Mo) and mineral (P, S, Ca, Mg, K, Na, Zn, Fe, Cu, Mn) elements were systematically investigated in botanically and geologically defined royal jelly samples. In addition, concentrations of 14 trace elements were measured in the associated honey samples--honey being the precursor of royal jelly. Concentrations of K, Na, Mg, Ca, P, S, Cu, Fe, Zn, Al, Ba and Sr in royal jelly were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), while concentrations of Bi, Cd, Hg, Pb, Sn, Te, Tl, W, Sb, Cr, Mn, Ni, Ti, V, Co and Mo in royal jelly were determined by double focusing magnetic sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). In the honey samples, trace and mineral element concentrations strongly depended on botanical and geological origin, and substantial variation was found. In contrast, the concentrations of trace and mineral elements were highly constant in the associated royal jelly samples. The most important results were the homeostatic adjustments of trace and mineral element concentrations in royal jelly. This effect was evidently produced in the endocrine glands of nurse bees, which are adapted for needs of bee larvae. In conclusion, this research yielded a surprising and completely new finding--that royal jelly, as a form of lactation on the insect level, shows the same homeostatic adjustment as mammalian and human breast milk.     

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。     

意蜂王浆生产性能形态学遗传标记的研究

测定19只工蜂头部咽下腺的两条腺体长度和重量,经生统t检验,发现同一工蜂的左、右两条咽下腺长度、重量差异都不显著（P＜0.05）。测定11群 "浙农大1号"意蜂(Apis mellifera ligustica)的330只工蜂的头重、体重、头体重比、咽下腺小囊数和咽下腺长度。在2001年3月30日至4月26日浙江淳安的油菜和紫云英花期测定试验蜂群的王浆产量,分析工蜂头重、头体重比、咽下腺小囊数和咽下腺长度与每千克蜂的王浆产量之间的相关性,发现咽下腺长度与王浆产量的相关性最大,小囊数次之,咽下腺长度可作为意蜂王浆生产性能较理想的形态学遗传标记。 Abstract:The lengths of hypopharyngeal glands (HG) from the left and right side were determined in 19 workers of honeybee(Apis mellifera ligustica).There were no significant differences (P＜0.05) in length between the left and the right in one worker′s hypopharyngeal gland.Three hundred and thirty workers were collected from eleven colonies of "ZND No.1" Italian honeybee(Apis mellifera ligustica) respectively.Head weight,body weight,ratio between head weight and body weight,bursa number and length of hypopharyngeal gland(HG)were tested in these samples.Royal jelly productions were determined during the flowing period of rape and Chinese milk retch from March 30 to April 26 in Chun′an County of Zhejiang Province in 2001.The correlation analysis between royal jelly production and head weight,ratio between head weight and body weight,bursa number of HG,and length of HG were conducted.The correlation coefficient between royal jelly production and length of HG was the largest.The correlation coefficient between royal jelly production and bursa number was the second.It was suggested that the length of HG could be used as one of genetic markers for the production performance of royal jelly.     

Pollen analysis as a means to determine the geographical origin of royal jelly

In this study we investigated the use of pollen analysis as a method to determine the geographical origin of royal jelly. We recorded the pollen flora sampled by bees using pollen traps for two consecutive years and we also collected and examined royal jelly samples from the same apiary. For royal jelly production, bees mainly used the freshly collected pollen. All major pollen types that were recorded in the area using pollen traps were also detected in the royal jelly samples. Thus, pollen analysis can be used as a method to determine the geographical origin of royal jelly.     

Synthesis of insect pheromones: Improved method for the preparation of queen substance and royal jelly of honeybees Apis mellifera

The biological activity of pheromones stems from various applications, especially in the area of pest control and insect monitoring; however, the quantity of pure pheromones available from natural sources is often extremely limited, because individual insects contain only minute amounts of pheromone. As our contribution to the field of pheromone synthesis, here we present a novel approach for the preparation of two known pheromones, namely queen substance and royal jelly of honeybees Apis mellifera. Our method is based on the original applications of lithiated α-alkenylphosphoramido anions as excellent synthetic equivalents of homoenolate anions.     

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.     

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.     

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.     

Effect of royal jelly on bisphenol A-induced proliferation of human breast cancer cells

Royal jelly is known as a functional food containing many useful minerals. In this study, we found an anti-environmental estrogen activity of royal jelly. Bisphenol A (BPA) is an environmental estrogen that stimulates proliferation of human breast cancer MCF-7 cells. Royal jelly inhibited the growth-promoting effect of BPA on MCF-7 cells, even though it did not affect the proliferation of cells in the absence of BPA. In addition, this inhibiting effect of royal jelly was heat-stable.     

蜜蜂蜂王浆主蛋白(MRJPs)的研究进展

蜜蜂的蜂王浆主蛋白具有为蜂王和幼虫提供营养、影响蜂群社会行为及调节个体生理机能等作用,作为蜂王浆的主要成分对其他机体也可产生多方面的生物学功能。因此,近年来蜂王浆主蛋白的相关研究备受关注。本文针对蜂王浆主蛋白的发现、种类、功能、系统进化及其基因表达情况进行了系统综述。     

Molecular and biochemical characterization of the major royal jelly protein in bumblebees suggest a non-nutritive function

Honeybee queens are generated on purpose by extensive feeding with a glandular secretion termed royal jelly. Major royal jelly proteins (MRJPs) are the dominant proteinaceous component of royal jelly. One of them, MRJP1, was found to play a central role in honeybee queen development. Genes encoding MRJPs were reported to originate from a single originator, and several of them have evolved nutritive function. Phylogenetic analysis provides evidence that the same originator has multiplied independently in Nasonia and ant lineages. Here we show that bumblebees represent a transition species preserving a single-copy pre-multiplication stage of MRJP evolution. By exploring the single-copy BtRJPL gene, we found striking similarities with MRJPs of the honeybee such as gene structure and expression regulation. At the same time it turned out that BtRJPL does not fulfill criteria for functioning as a nutritive protein. Instead we found evidence that BtRJPL is involved in food digestion or modification, which appears to be the original MRJP function, at least in this lineage. Thus, the evolutionary pattern of MRJPs in hymenopterans constitutes an excellent example of a functional diversification combined with the origin of new properties followed by intensive gene duplication events.     

Ethanol‐soluble proteins from the royal jelly of Xinjiang black bees

Royal jelly is a nutritious food that has beneficial effects to human health. However, the functional substances remain unclear. Herein, we fractioned the royal jelly proteins of Xinjing black bees according to the Osboren method. Two main proteins from the ethanol‐soluble fraction were purified and identified. RJG‐1 was determined as glucosylceramidase, and RJG‐2 was major royal jelly protein 1 (MRJP1). RJG‐1 showed potent cytotoxicity toward various mammalian cells, and caused quick disruption of cell membranes. With glucosylceramidase activity, RJG‐1 may degrade the glucosylceramide of the cell membranes and disrupt the membrane structure, thereby resulting in cell necrosis. This study extends our knowledge about the composition and function of royal jelly, and is significant for the application of royal jelly.     

Identification of a Royal Jelly Glycoprotein That Carries Unique Complex-Type N-Glycans Harboring the T-Antigen (Galβ1-3GalNAc) Unit

In this study, we identified a royal jelly glycoprotein (RJG) that carries a unique complex-type N-glycans harboring the T-antigen (Galβ1-3GalNAc) unit. The amino acid sequence of the tryptic glycopeptide harboring the T-antigen unit was G-E-S-L-X-K (X might be glycosylated Asn), confirmed in the major royal jelly glycoprotein 1 (MRJP1), which is also expressed in the mushroom body of the honeybee brain.