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Honey bee larval food jelly is a secretion of the hypopharyngeal and mandibular glands of young worker bees that take care of the growing brood in the hive. Food jelly is fed to all larvae (workers, drones and queens) and as royal jelly to the queen bee for her entire life. Up to 18% of the food jelly account for proteins the majority of which belongs to the major royal jelly protein (MRJP) family. These proteins are produced in the hypopharyngeal glands at a pH value of 7.0. Before being fed to the larvae, they are mixed with the fatty acids secreted by the mandibular glands of the worker bees resulting at a pH of 4.0 in the food jelly. Thus, MRJPs are exposed to a broad pH range from their site of synthesis to the actual secreted larval food. We therefore determined the pH-dependent stability of MRJP1, MRJP2 and MRJP3 purified from royal jelly using differential scanning fluorimetry. All MRJPs were much more stable at acidic pH values compared to neutral ones with all proteins showing highest stability at pH 4.0 or 4.5, the native pH of royal jelly.

The life-prolonging effects of antioxidants have long entered popular culture, but the scientific community still debates whether free radicals and the resulting oxidative stress negatively affect longevity. Social insects are intriguing models for analysing the relationship between oxidative stress and senescence because life histories differ vastly between long-lived reproductives and the genetically similar but short-lived workers. Here, we present the results of an experiment on the accumulation of oxidative damage to proteins, and a comparative analysis of the expression of 20 selected genes commonly involved in managing oxidative damage, across four species of social insects: a termite, two bees and an ant. Although the source of analysed tissue varied across the four species, our results suggest that oxidative stress is a significant factor in senescence and that its manifestation and antioxidant defenses differ among species, making it difficult to find general patterns. More detailed and controlled investigations on why responses to oxidative stress may differ across social species may lead to a better understanding of the relations between oxidative stress, antioxidants, social life history and senescence.This article is part of the theme issue ‘Ageing and sociality: why, when and how does sociality change ageing patterns?'

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The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/ Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40–95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70–95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Honey bee (Apis spp.) royal jelly, a glandular secretion used to raise young larvae to future queens, has long been considered merely as food. Since queen larvae are raised upside down in their vertically oriented queen cells, royal jelly also needs to adhere the larvae to the cell ceiling to prevent the prospective queen from dropping out. This is exactly where the native acidic pH of royal jelly comes into play: only at a pH of 4.0 is royal jelly viscous enough to hold the larvae in their cells. We here show with the help of electron microscopy that royal jelly possesses a complex tissue-like organization at pH 4.0 which is similar to the dense extracellular matrix of animals providing structural support. The main structural elements at pH 4.0 are proteinaceous fibril bundles, embedded in a fibrillary net, that seem to be bunched in electron-dense structures, potential sites of fibril overlap and cross-linking. At an exogenously induced increased royal jelly pH of 7.0, these fibrillary structures are largely destroyed. This is when royal jelly viscosity decreases and holding the queen larvae in place is no longer guaranteed.

The western honeybee, Apis mellifera is a prominent model organism in the field of sociogenomics and a recent upgrade substantially improved annotations of the reference genome. Nevertheless, genome assemblies based on short-sequencing reads suffer from problems in regions comprising e.g. multi-copy genes. We used single-molecule nanopore-based sequencing with extensive read-lengths to reconstruct the organization of the major royal jelly protein (mrjp) region in three species of the genus Apis. Long-amplicon sequencing provides evidence for lineage-specific evolutionary fates of Apis mrjps. Whereas the most basal species, A. florea, seems to encode ten mrjps, different patterns of gene loss and retention were observed for A. mellifera and A. dorsata. Furthermore, we show that a previously reported pseudogene in A. mellifera, mrjp2-like, is an assembly artefact arising from short read sequencing.

Honey bee larval food jelly is a secretion of the hypopharyngeal and mandibular glands of young worker bees that take care of the growing brood in the hive. Food jelly is fed to all larvae (workers, drones and queens) and as royal jelly to the queen bee for her entire life. Up to 18% of the food jelly account for proteins the majority of which belongs to the major royal jelly protein (MRJP) family. These proteins are produced in the hypopharyngeal glands at a pH value of 7.0. Before being fed to the larvae, they are mixed with the fatty acids secreted by the mandibular glands of the worker bees resulting at a pH of 4.0 in the food jelly. Thus, MRJPs are exposed to a broad pH range from their site of synthesis to the actual secreted larval food. We therefore determined the pH-dependent stability of MRJP1, MRJP2 and MRJP3 purified from royal jelly using differential scanning fluorimetry. All MRJPs were much more stable at acidic pH values compared to neutral ones with all proteins showing highest stability at pH 4.0 or 4.5, the native pH of royal jelly.

Royal jelly (RJ) is a beehive product with a complex composition, major royal jelly proteins (MRJPs) being the most abundant proteins. Cell culture and animal studies suggest various biological activities for the full-length/native MRJPs. In the field of apitherapy, it is assumed that MRJPs can positively affect human health. However, whenever RJ is administered orally, the availability for assimilation in the gastrointestinal tract is a prerequisite for MRJPs to have any effect on humans. We here show that MRJPs vary in resistance to pepsin digestion with MRJP2 being most stable and still present as full-length protein after 24 h of digestion. In the intestinal phase, using trypsin and chymotrypsin, MRJPs are rapidly digested with MRJP2 again showing longest stability (40 min), suggesting that MRJPs can reach the small intestine as full-length proteins but then have to be resorbed quickly if full-length proteins are to fulfill any biological activity.

The female sex in honeybees (Apis spp.) comprises a reproductive queen and a sterile worker caste. Nurse bees feed all larvae progressively with a caste-specific food jelly until the prepupal stage. Only those larvae that are exclusively fed a large amount of royal jelly (RJ) develop into queens [1]. RJ is a composite secretion of two specialized head glands: the mandibular glands, which produce mainly fatty acids [2], and the hypopharyngeal glands, which contribute proteins, primarily belonging to the major royal jelly protein (MRJP) family [3]. Past research on RJ has focused on its nutritional function and overlooked its central role with regard to the orientation of the larva in the royal brood cell. Whereas workers are reared in the regular horizontal cells of the comb, the queen cells are specifically built outside of the normal comb area to accommodate for the larger queen [4, 5]. These cells hang freely along the bottom of the comb and are vertically oriented, opening downward [6]. Queen larvae are attached by their RJ diet to the cell ceiling. Thus, the physical properties of RJ are central to successful retention of larvae in the cell. Here, we show that the main protein of RJ (MRJP1) polymerizes in complex with another protein, apisimin, into long fibrous structures that build the basis for the high viscosity of RJ to hold queen larvae on the RJ surface.

Royalactin is not a royal making of a queen Anja Buttstedt;Christian Ihling;Markus Pietzsch;Robin Moritz; Nature

SummaryRoyal jelly (RJ) is secreted by worker bees of Apis mellifera to feed the queen larvae. Believed to be the longevity factor of queen honey bees and due to its variety of health promoting ingredients, RJ is used worldwide as dietary supplement, health product and in cosmetics. To reach best possible effects, application of high quality RJ is absolutely required. Here we show, based on physicochemical properties and antioxidant activity, a comparison between fresh, local RJ samples obtained directly from beekeepers and commercial ones. Analyses revealed a higher content of 10-hydroxy-2-decenoic acid, a quality parameter of RJ, for local compared to commercial RJ. Furthermore, determined ferric reducing antioxidant power (FRAP) correlated with radical scavenging activity (DPPH) and total polyphenols was significantly higher in local RJ samples. Therefore, our results indicate a higher quality for local RJ compared to commercial.

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.

In vitro amyloid formation has been suggested to be a common property of any polypeptide chain depending on particular environmental conditions although in vivo amyloid fibril formation can be promoted by point mutations or triplet expansions. Here, we explored the influence of agitation on fibril formation of amyloidogenic alanine segments fused to Cold Shock Protein B (CspB) of Bacillus subtilis. While without agitation fibril formation was clearly dependent on the presence of an amyloidogenic alanine segment, fibril formation was independent of the amyloidogenic segment under agitation. Agitation even led to fibrillation of native CspB lacking the amyloidogenic segment. Furthermore, agitation not only influenced the kinetics of fibril formation, but also resulted in completely different fibril morphologies. These results indicate that experimental conditions can alter the region that undergoes a conformational change during in vitro fibrillation. Moreover, the data show that deductions from in vitro assays on in vivo fibril formation mechanisms are afflicted with a certain degree of uncertainty and therefore need to be cautiously discussed.

Reproduction has been shown to be costly for survival in a wide diversity of taxa. The resulting trade-off, termed the reproduction-survival trade-off, is thought to be one of the most fundamental forces of life-history evolution. In insects the pleiotropic effect of juvenile hormone (JH), antagonistically regulating reproduction and pathogen resistance, is suggested to underlie this phenomenon. In contrast to the majority of insects, reproductive individuals in many eusocial insects defy this trade-off and live both long and prosper. By remodelling the gonadotropic effects of JH in reproductive regulation, the queens of the long-lived black garden ant Lasius niger (living up to 27 years), have circumvented the reproduction-survival trade off enabling them to maximize both reproduction and pathogen resistance simultaneously. In this study we measure fertility, vitellogenin gene expression and protein levels after experimental manipulation of hormone levels. We use these measurements to investigate the mechanistic basis of endocrinological role remodelling in reproduction and determine how JH suppresses reproduction in this species, rather then stimulating it, like in the majority of insects. We find that JH likely inhibits three key aspects of reproduction both during vitellogenesis and oogenesis, including two previously unknown mechanisms. In addition, we document that juvenile hormone, as in the majority of insects, has retained some stimulatory function in regulating vitellogenin expression. We discuss the evolutionary consequences of this complex regulatory architecture of reproduction in L. niger, which might enable the evolution of similar reproductive phenotypes by alternate regulatory pathways, and the surprising flexibility regulatory role of juvenile hormone in this process.

Die Wachstumsfaktoren der Cystin-Knoten-Familie haben eine charakteristische Anordnung der Disulfidbrucken gemeinsam. Die sezernierten Proteine werden als Pra-Pro-Formen mit umfangreichen Pro-Domanen synthetisiert, deren Funktionen bislang kaum verstanden sind.

Summary The female sex in honeybees ( Apis spp. ) comprises a reproductive queen and a sterile worker caste. Nurse bees feed all larvae progressively with a caste-specific food jelly until the prepupal stage. Only those larvae that are exclusively fed a large amount of royal jelly (RJ) develop into queens [1]. RJ is a composite secretion of two specialized head glands: the mandibular glands, which produce mainly fatty acids [2], and the hypopharyngeal glands, which contribute proteins, primarily belonging to the major royal jelly protein (MRJP) family [3]. Past research on RJ has focused on its nutritional function and overlooked its central role with regard to the orientation of the larva in the royal brood cell. Whereas workers are reared in the regular horizontal cells of the comb, the queen cells are specifically built outside of the normal comb area to accommodate for the larger queen [4, 5]. These cells hang freely along the bottom of the comb and are vertically oriented, opening downward [6]. Queen larvae are attached by their RJ diet to the cell ceiling. Thus, the physical properties of RJ are central to successful retention of larvae in the cell. Here, we show that the main protein of RJ (MRJP1) polymerizes in complex with another protein, apisimin, into long fibrous structures that build the basis for the high viscosity of RJ to hold queen larvae on the RJ surface.

Pro-forms of growth factors have received increasing attention since it was shown that they can affect both the maturation and functions of mature growth factors. Here, we assessed the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor beta (TGFbeta)/BMP superfamily. The role of the 263 amino acids of the pro-peptide is currently unclear. In order to obtain an insight into the function of the pro-form (proBMP-2), the ability of proBMP-2 to induce alkaline phosphatase (AP), a marker enzyme for cells differentiating into osteoblasts, was tested. Interestingly, in contrast to mature BMP-2, proBMP-2 did not lead to induction of AP. Instead, proBMP-2 inhibited the induction of AP by BMP-2. This result raised the question of whether proBMP-2 may compete with mature BMP-2 for receptor binding. ProBMP-2 was found to bind to the purified extracellular ligand binding domain (ECD) of BMPR-IA, a high-affinity receptor for mature BMP-2, with a similar affinity as mature BMP-2. Binding of proBMP-2 to BMPR-IA was confirmed in cell culture by cross-linking proBMP-2 to BMPR-IA presented on the cell surface. In contrast to this finding, proBMP-2 did not bind to the ECD of BMPR-II. ProBMP-2 also differed from BMP-2 in its capacity to induce p38 and Smad phosphorylation. The data presented here suggest that the pro-domain of BMP-2 can alter the signalling properties of the growth factor by modulating the ability of the mature part to interact with the receptors.