The Effect of Diet on the Composition and Stability of Proteins Secreted by Honey Bees in Honey

Honey proteins are essential bee nutrients and antimicrobials that protect honey from microbial spoilage. The majority of the honey proteome includes bee-secreted peptides and proteins, produced in specialised glands; however, bees need to forage actively for nitrogen sources and other basic elements of protein synthesis. Nectar and pollen of different origins can vary significantly in their nutritional composition and other compounds such as plant secondary metabolites. Worker bees producing and ripening honey from nectar might therefore need to adjust protein secretions depending on the quality and specific contents of the starting material. Here, we assessed the impact of different food sources (sugar solutions with different additives) on honey proteome composition and stability, using controlled cage experiments. Honey-like products generated from sugar solution with or without additional protein, or plant secondary metabolites, differed neither in protein quality nor in protein quantity among samples. Storage for 4 weeks prevented protein degradation in most cases, without differences between food sources. The honey-like product proteome included several major royal jelly proteins, alpha-glucosidase and glucose oxidase. As none of the feeding regimes resulted in different protein profiles, we can conclude that worker bees may secrete a constant amount of each bee-specific protein into honey to preserve this highly valuable hive product

In the battle of the disease: a transcriptomic analysis of European foulbrood-diseased larvae of the Western honey bee (Apis mellifera)

Background European foulbrood is a significant bacterial brood disease of Apis sp. and can cause severe and devastating damages in beekeeping operations. Nevertheless, the epidemiology of its causative agent Melissococcus plutonius has been begun to uncover but the underlying mechanisms of infection and cause of disease still is not well understood. Here, we sought to provide insight into the infection mechanism of EFB employing RNAseq in in vitro reared Apis mellifera larvae of two developmental stages to trace transcriptional changes in the course of the disease, including Paenibacillus alvei secondary infected individuals. Results In consideration of the progressing development of the larva, we show that infected individuals incur a shift in metabolic and structural protein-encoding genes, which are involved in metabolism of crucial compounds including all branches of macronutrient metabolism, transport protein genes and most strikingly chitin and cuticle associated genes. These changes underpin the frequently observed developmental retardation in EFB disease. Further, sets of expressed genes markedly differ in different stages of infection with almost no overlap. In an earlier stage of infection, a group of regulators of the melanization response cascade and complement component-like genes, predominantly C-type lectin genes, are up-regulated while a differential expression of immune effector genes is completely missing. In contrast, late-stage infected larvae up-regulated the expression of antimicrobial peptides, lysozymes and prominent bacteria-binding haemocyte receptor genes compared to controls. While we clearly show a significant effect of infection on expressed genes, these changes may partly result from a shift in expression timing due to developmental alterations of infection. A secondary infection with P. alvei elicits a specific response with most of the M. plutonius associated differential immune effector gene expression missing and several immune pathway genes even down-regulated. Conclusion We conclude that with progressing infection diseased individuals undergo a systemic response with a change of metabolism and their activated immune defence repertoire. Moreover, larvae are capable of adjusting their response to a secondary invasion in late stage infections

Changes in chemical cues of Melissococcus plutonius infected honey bee larvae

International audienceEuropean foulbrood (EFB), caused by Melissococcus plutonius, is a globally distributed bacterial brood disease affecting Apis mellifera larvae. There is some evidence, even if under debate, that spreading of the disease within the colony is prevented by worker bees performing hygienic behaviour, including detection and removal of infected larvae. Olfactory cues (brood pheromones, signature mixtures, diagnostic substances) emitted by infected individuals may play a central role for hygienic bees to initiate the disease-specific behaviour. However, the mechanisms of cue detection and brood removal, causing hygienic behaviour in EFB affected colonies, are poorly understood. Here, coupled gas chromatography-mass spectrometry (GC-MS) was used to detect disease-specific substances, changes in cuticular hydrocarbon (CHC) profiles, and brood ester pheromones (BEPs) of honey bee larvae artificially infected with M. plutonius. Although no diagnostic substances were found in significant quantities, discriminant analysis revealed specific differences in CHC and BEP profiles of infected and healthy larvae. β-Ocimene, a volatile brood pheromone related to starvation and hygienic behaviour, was present in all larvae with highest quantities in healthy young larvae; whereas oleic acid, a non-volatile necromone, was present only in old infected larvae. Furthermore, γ-octalactone (newly discovered in A. mellifera in this study) was detectable in trace amounts only in infected larvae. We propose that the deviation from the olfactory profile of healthy brood is supposed to trigger hygienic behaviour in worker bees. To confirm the relevance of change in the chemical bouquet (CHCs, BEPs, γ-octalactone, etc.), a field colony bioassay is needed, using healthy brood and hygienic bees to determine if bouquet changes elicit hygienic behaviour

Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis

Nosema ceranae is an intracellular microsporidian parasite that infects epithelial cells of the honey bee (Apis mellifera) midgut. Previous studies have shown that Nosema may alter cell renewal and apoptosis in honey bees. We found that the amount of apoptotic cells progressively declines from the anterior towards posterior regions of the midgut in Nosema-infected sensitive bees. There was no such pattern in the infected Nosema tolerant honeybees and controls. These data provide additional evidence that N. ceranae appears to alter apoptosis in its host cells for its own advantage

<em>Nosema</em> Tolerant Honeybees (<em>Apis mellifera</em>) Escape Parasitic Manipulation of Apoptosis

International audienceApoptosis is not only pivotal for development, but also for pathogen defence in multicellular organisms. Although numerous intracellular pathogens are known to interfere with the host’s apoptotic machinery to overcome this defence, its importance for host-parasite coevolution has been neglected. We conducted three inoculation experiments to investigate in the apoptotic respond during infection with the intracellular gut pathogen Nosema ceranae, which is considered as potential global threat to the honeybee (Apis mellifera) and other bee pollinators, in sensitive and tolerant honeybees. To explore apoptotic processes in the gut epithelium, we visualised apoptotic cells using TUNEL assays and measured the relative expression levels of subset of candidate genes involved in the apoptotic machinery using qPCR. Our results suggest that N. ceranae reduces apoptosis in sensitive honeybees by enhancing inhibitor of apoptosis protein-(iap)-2 gene transcription. Interestingly, this seems not be the case in Nosema tolerant honeybees. We propose that these tolerant honeybees are able to escape the manipulation of apoptosis by N. ceranae, which may have evolved a mechanism to regulate an anti-apoptotic gene as key adaptation for improved host invasion

Relative expression (mean ± s.e.) of candidate genes important for apoptosis in <i>Nosema</i> infected honeybees.

<p><i>Nosema</i> sensitive (SN, solid circles) and tolerant (TN, solid squares) honeybees infected with 10⁵<i>N</i>. <i>ceranae</i> spores, and their controls uninfected (SC, open circles and TC, open squares), were sampled at 1 day (green) and 6 days (blue) after inoculation. The genes JNK/<i>bsk</i> (Jun N–terminal kinase/ <i>basket</i>), <i>p53</i> (<i>tumor protein p53-like</i>), <i>iap–2</i> (<i>inhibitor of apoptosis protein 2;</i> predicted homologous gene to <i>Diap–1</i> in <i>D</i>. <i>melanogaster</i>), <i>casp–2</i> (<i>caspase–2–like; homologous gene to Dcp–1</i>), <i>casp–10</i> (<i>caspase–10–like; homologous gene to Dredd</i>) were predicted from <i>Drosophila melanogaster</i>. Sample sizes are ranging between six and ten pools of three individual honeybee midguts (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140174#pone.0140174.s003" target="_blank">S3 Table</a>). Significance between treatment groups ***, <i>P</i> < 0.001.</p