RESUMO
Adult workers of Western honey bees (Apis mellifera L.) acquire sterols from their pollen diet. These food sterols are transported by the hemolymph to peripheral tissues such as the mandibular and the hypopharyngeal glands in the worker bees' heads that secrete food jelly which is fed to developing larvae. As sterols are obligatory components of biological membranes and essential precursors for molting hormone synthesis in insects, they are indispensable to normal larval development. Thus, the study of sterol delivery to larvae is important for a full understanding of honey bee larval nutrition and development. Whereas hypopharyngeal glands only require sterols for their membrane integrity, mandibular glands add sterols, primarily 24-methylenecholesterol, to its secretion. For this, sterols must be transported through the glandular epithelial cells. We have analyzed for the first time in A. mellifera the expression of genes which are involved in intracellular movement of sterols. Mandibular and hypopharyngeal glands were dissected from newly emerged bees, 6-day-old nurse bees that feed larvae and 26-day-old forager bees. The expression of seven genes involved in intracellular sterol metabolism was measured with quantitative real-time PCR. Relative transcript abundance of sterol metabolism genes was significantly influenced by the age of workers and specific genes but not by gland type. Newly emerged bees had significantly more transcripts for six out of seven genes than older bees indicating that the bulk of the proteins needed for sterol metabolism are produced directly after emergence.
Assuntos
Homeostase , Proteínas de Insetos , Esteróis , Abelhas/genética , Animais , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Esteróis/metabolismo , Hipofaringe/metabolismo , Regulação da Expressão Gênica , Larva/metabolismo , Larva/genéticaRESUMO
Quantitative real-time polymerase chain reaction (qPCR) is a method widely used to determine changes and differences in gene expression. As target gene expression is most often quantified relative to the expression of reference genes, the validation of suitable reference genes is of critical importance. In practice, however, such validation might not be thoroughly conducted if the same species and the same tissue or body parts are used for qPCR experiments. Here we show, that qPCR reference genes published for workers of European honey bee (Apis mellifera) subspecies fail to be stably expressed in workers of the African subspecies Apis mellifera scutellata. This is the case even when the sampled workers are in the same life stage, the same organ was dissected and the same reagents were used. Thus, reference genes need to be thoroughly re-tested before they can be used as suitable references even when the only thing that changes is the subspecies used.
RESUMO
Honey bee (Apis mellifera) workers feed their larvae with food jelly that is secreted by specialized glands in their heads - the hypopharyngeal and the mandibular glands. Food jelly contains all the nutrients the larvae need to develop into adult honey bees, including essential dietary sterols. The main sterol in food jelly, 24-methylenecholesterol (24MC), is pollen-derived and delivered in food jelly to the larvae in a complex with two proteins, major royal jelly protein 1 (MRJP1) and apisim. Whereas the proteins are synthesized in the hypopharyngeal glands, the sterol-secreting gland has not been identified. We here identified the mandibular glands as sterol-secreting gland for food jelly production by direct detection of the four main honey bee sterols (24MC, campesterol, ß-sitosterol and isofucosterol). Furthermore, 24MC seems to be specifically enriched in the mandibular glands, thereby ensuring that food jelly contains the amounts of 24MC necessary for complex formation with MRJP1 and apisimin.
Assuntos
Proteínas de Insetos , Esteróis , Abelhas , Animais , Larva/metabolismo , Proteínas de Insetos/metabolismo , NutrientesRESUMO
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?'
Assuntos
Envelhecimento , Antioxidantes/metabolismo , Formigas/fisiologia , Abelhas/fisiologia , Isópteros/fisiologia , Estresse Oxidativo , Animais , Especificidade da EspécieRESUMO
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.
Assuntos
Especiação Genética , Genoma de Inseto , Interações Hospedeiro-Parasita/genética , Himenópteros/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Elementos de DNA Transponíveis , Feminino , Dosagem de Genes , Glicoproteínas/genética , Herbivoria/genética , Imunidade/genética , Proteínas de Insetos/genética , Masculino , Família Multigênica , Receptores Odorantes/genética , Comportamento Social , Visão Ocular/genéticaRESUMO
The genome of the western honeybee (Apis mellifera) harbors nine transcribed major royal jelly protein genes (mrjp1-9) which originate from a single-copy precursor via gene duplication. The first MRJP was identified in royal jelly, a secretion of the bees' hypopharyngeal glands that is used by young worker bees, called nurses, to feed developing larvae. Thus, MRJPs are frequently assumed to mainly have functions for developing bee larvae and to be expressed in the food glands of nurse bees. In-depth knowledge on caste- and age-specific role and abundance of MRJPs is missing. We here show, using combined quantitative real-time PCR with quantitative mass spectrometry, that expression and protein amount of mrjp1-5 and mrjp7 show an age-dependent pattern in worker's hypopharyngeal glands as well as in brains, albeit lower relative abundance in brains than in glands. Expression increases after hatching until the nurse bee period and is followed by a decrease in older workers that forage for plant products. Mrjp6 expression deviates considerably from the expression profiles of the other mrjps, does not significantly vary in the brain, and shows its highest expression in the hypopharyngeal glands during the forager period. Furthermore, it is the only mrjp of which transcript abundance does not correlate with protein amount. Mrjp8 and mrjp9 show, compared to the other mrjps, a very low expression in both tissues. Albeit mrjp8 mRNA was detected via qPCR, the protein was not quantified in any of the tissues. Due to the occurrence of MRJP8 and MRJP9 in other body parts of the bees, for example, the venom gland, they might not have a hypopharyngeal gland- or brain-specific function but rather functions in other tissues. Thus, mrjp1-7 but not mrjp8 and mrjp9 might be involved in the regulation of phenotypic plasticity and age polyethism in worker honeybees.
RESUMO
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.
Assuntos
Abelhas/metabolismo , Glicoproteínas/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Ácidos Graxos/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Hipofaringe/metabolismo , Larva/crescimento & desenvolvimento , Masculino , Estabilidade ProteicaRESUMO
One of the first tasks of worker honey bees (Apis mellifera) during their lifetime is to feed the larval offspring. In brief, young workers (nurse bees) secrete a special food jelly that contains a large amount of unique major royal jelly proteins (MRJPs). The regulation of mrjp gene expression is not well understood, but the large upregulation in well-fed nurse bees suggests a tight repression until, or a massive induction upon, hatching of the adult worker bees. The lipoprotein vitellogenin, the synthesis of which is regulated by the two systemic hormones 20-hydroxyecdysone and juvenile hormone, is thought to be a precursor for the production of MRJPs. Thus, the regulation of mrjp expression by the said systemic hormones is likely. This study focusses on the role of 20-hydroxyecdysone by elucidating its effect on mrjp gene expression dynamics. Specifically, we tested whether 20-hydroxyecdysone displayed differential effects on various mrjps. We found that the expression of the mrjps (mrjp1â»3) that were finally secreted in large amounts into the food jelly, in particular, were down regulated by 20-hydroxyecdysone treatment, with mrjp3 showing the highest repression value.
RESUMO
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.
Assuntos
Ácidos Graxos/química , Ácidos Graxos/metabolismo , Trato Gastrointestinal/metabolismo , Animais , Abelhas , Digestão , Trato Gastrointestinal/química , Humanos , Cinética , ProteóliseRESUMO
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.
Assuntos
Abelhas/crescimento & desenvolvimento , Ácidos Graxos/metabolismo , Gravitação , Proteínas de Insetos/metabolismo , Reprodução , Comportamento Social , Animais , Abelhas/fisiologia , Ácidos Graxos/química , Feminino , Larva , ViscosidadeRESUMO
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.
Assuntos
Abelhas/metabolismo , Ácidos Graxos/genética , Genômica , Família Multigênica , Animais , Abelhas/genética , Evolução Molecular , Feminino , Análise de Sequência de DNARESUMO
Honeybee colonies (Apis mellifera) serve as attractive hosts for a variety of pathogens providing optimal temperatures, humidity, and an abundance of food. Thus, honeybees have to deal with pathogens throughout their lives and, even as larvae they are affected by severe brood diseases like the European Foulbrood caused by Melissococcus plutonius. Accordingly, it is highly adaptive that larval food jelly contains antibiotic compounds. However, although food jelly is primarily consumed by bee larvae, studies investigating the antibiotic effects of this jelly have largely concentrated on bacterial human diseases. In this study, we show that royal jelly fed to queen larvae and added to the jelly of drone and worker larvae, inhibits not only the growth of European Foulbrood-associated bacteria but also its causative agent M. plutonius. This effect is shown to be caused by the main protein (major royal jelly protein 1) of royal jelly.
Assuntos
Antibacterianos/farmacologia , Enterococcaceae/crescimento & desenvolvimento , Ácidos Graxos/análise , Ácidos Graxos/farmacologia , Glicoproteínas/farmacologia , Proteínas de Insetos/farmacologia , Animais , Abelhas/metabolismo , Enterococcaceae/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Abelhas/genética , Abelhas/fisiologia , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Insetos/classificação , Proteínas de Insetos/metabolismo , Animais , Ácidos Graxos/química , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimentoRESUMO
To date five different theories compete in explaining the biological mechanisms of senescence or ageing in invertebrates. Physiological, genetical, and environmental mechanisms form the image of ageing in individuals and groups. Social insects, especially the honeybee Apis mellifera, present exceptional model systems to study developmentally related ageing. The extremely high phenotypic plasticity for life expectancy resulting from the female caste system provides a most useful system to study open questions with respect to ageing. Here, we used long-lived winter worker honeybees and measured transcriptional changes of 14 antioxidative enzyme, immunity, and ageing-related (insulin/insulin-like growth factor signaling pathway) genes at two time points during hibernation. Additionally, worker bees were challenged with a bacterial infection to test ageing- and infection-associated immunity changes. Gene expression levels for each group of target genes revealed that ageing had a much higher impact than the bacterial challenge, notably for immunity-related genes. Antimicrobial peptide and antioxidative enzyme genes were significantly upregulated in aged worker honeybees independent of bacterial infections. The known ageing markers vitellogenin and IlP-1 were opposed regulated with decreasing vitellogenin levels during ageing. The increased antioxidative enzyme and antimicrobial peptide gene expression may contribute to a retardation of senescence in long-lived hibernating worker honeybees.
Assuntos
Envelhecimento/fisiologia , Antioxidantes/metabolismo , Abelhas/fisiologia , Imunidade Inata , Longevidade , Modelos Biológicos , Vitelogeninas/metabolismo , Animais , Feminino , Fenótipo , Estações do AnoRESUMO
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.
RESUMO
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.
Assuntos
Alanina/metabolismo , Amiloide/química , Amiloide/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Fragmentos de Peptídeos/metabolismo , Agitação Psicomotora , Proteínas Recombinantes de Fusão/metabolismo , Alanina/genética , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , TermodinâmicaRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of specific muscles. OPMD is caused by extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Insoluble nuclear inclusions form in diseased muscles. We have generated a Drosophila model of OPMD that recapitulates the features of the disorder. Here, we show that the antiprion drugs 6-aminophenanthridine (6AP) and guanabenz acetate (GA), which prevent formation of amyloid fibers by prion proteins in cell models, alleviate OPMD phenotypes in Drosophila, including muscle degeneration and nuclear inclusion formation. The large ribosomal RNA and its activity in protein folding were recently identified as a specific cellular target of 6AP and GA. We show that deletions of the ribosomal DNA locus reduce OPMD phenotypes and act synergistically with sub-effective doses of 6AP. In a complementary approach, we demonstrate that ribosomal RNA accelerates in vitro fibril formation of PABPN1 N-terminal domain. These results reveal the conserved role of ribosomal RNA in different protein aggregation disorders and identify 6AP and GA as general anti-aggregation molecules.
Assuntos
Guanabenzo/uso terapêutico , Distrofia Muscular Oculofaríngea/metabolismo , Fenantridinas/uso terapêutico , Proteína II de Ligação a Poli(A)/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Larva/metabolismo , Distrofia Muscular Oculofaríngea/tratamento farmacológico , Fenótipo , Doenças Priônicas/tratamento farmacológico , Dobramento de Proteína , RNA Ribossômico/metabolismoRESUMO
Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG(D)â=â12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG(D)â=â26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.
Assuntos
Alanina/química , Amiloide/química , Proteínas de Bactérias/química , Oligopeptídeos/química , Alanina/genética , Alanina/metabolismo , Amiloide/genética , Amiloide/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Dicroísmo Circular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica , Estabilidade Proteica , Desdobramento de Proteína/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Termodinâmica , Ureia/química , Ureia/farmacologiaRESUMO
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.