Honeybee (Apis mellifera) and bumblebee (Bombus terrestris) venom: analysis and immunological importance of the proteome

This PhD thesis consists of two main parts. The first part focused on further unraveling the venom composition of the honeybee (A. mellifera) and bumblebee (B. terrestris) by integrating genome, transcriptome and proteome information. Second, this work aimed to advance knowledge about the immunological implications of the venom proteome by investigating the allergenic properties of immunologically uncharacterized venom compounds and by analyzing the immunological relevance of allergen protein heterogeneity. In chapter 1, liquid chromatography-mass spectrometry was used to identify novel honeybee venom compounds, an approach which overcomes the issues of gel-based proteomics. In addition, we investigated if the antigen 5-like sequence, previously found by mining the honeybee genome, is expressed by the honeybee venom glands. Finally, we tried to confirm the presence of novel identified compounds and the antigen 5-like compound in the venom by analyzing their IgG4-reactivity using sera of immune beekeepers. In chapter 2 we explored the hidden honeybee venom proteome by integrating a combinatorial peptide ligand library venom pre-treatment with FTMS, while in chapter 3 the venom proteome of the European buff-tailed bumblebee, B. terrestris, was unraveled using an identical approach. Also genome information was used to obtain further insights in the venom composition of both species. In chapter 4 we investigated the nature of Api m 10 protein heterogeneity and explored its effect on IgE-reactivity using sera of honeybee venom allergic patients. In chapter 5, we evaluated the allergenic potential of the honeybee venom C1q-like and PVF1 proteins by analyzing IgE-reactivity and basophil activation

Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5

Standard methods for Apis mellifera venom research

Honey bees have a sting which allows them to inject venomous substances into the body of an opponent or attacker. As the sting originates from a modified ovipositor, it only occurs in the female insect, and this is a defining feature of the bee species that belong to a subclade of the Hymenoptera called Aculeata. There is considerable interest in bee venom research, primarily because of an important subset of the human population who will develop a sometimes life threatening allergic response after a bee sting. However, the use of honey bee venom goes much further, with alleged healing properties in ancient therapies and recent research. The present paper aims to standardize selected methods for honey bee venom research. It covers different methods of venom collection, characterization and storage. Much attention was also addressed to the determination of the biological activity of the venom and its use in the context of biomedical research, more specifically venom allergy. Finally, the procedure for the assignment of new venom allergens has been presented. Las abejas meliferas tienen un aguijon que les permite inyectar sustancias venenosas en el cuerpo de un oponente o atacante. El aguijon es un ovipositor modificado que solo se manifiesta en el insecto hembra, siendo este una caracteristica que define a las especies de abejas que pertenecen al subclado de himenopteros llamada Aculeata. Hay un interes considerable en la investigacion del veneno de abeja, principalmente debido a que un porcentaje importante de la poblacion humana desarrollara una respuesta alergica - a veces mortal - a la picadura de abeja. Sin embargo, el uso del veneno de la abeja melifera abarca mucho mas, con presuntas propiedades curativas en terapias antiguas e investigaciones recientes. El presente trabajo tiene como objetivo estandarizar metodos seleccionados para la investigacion del veneno de las abejas meliferas. Cubre diferentes metodos de recoleccion, caracterizacion y almacenamiento de veneno. Tambien se presto mucha atencion a la determinacion de la actividad biologica del veneno y su uso en el contexto de la investigacion biomedica, mas especificamente la alergia al veneno. Finalmente, se ha presentado el procedimiento para la asignacion de nuevos alergenos de veneno

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Honeybee Venom Proteome Profile of Queens and Winter Bees as Determined by a Mass Spectrometric Approach

Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings

In vitro diagnosis of Hymenoptera venom allergy and further development of component resolved diagnostics

For most people Hymenoptera stings result in transient and bothersome local inflammatory responses characterized by pain, itching, redness and swelling. In contrast, for those presenting an IgE-mediated allergic reaction, a re-sting may cause life-threatening reactions. In such patients, correct diagnosis is an absolute prerequisite for effective management, i.e. venom-specific immunotherapy. Generally, identification of the offending insect involves a detailed history along with quantification of venom-specific IgE antibodies and venom skin tests. Unfortunately, due to uncertainties associated with both tests, correct diagnosis is not always straightforward. This review summarizes the potentials and limitations of the various in vitro tests that are currently being used in the diagnosis of Hymenoptera venom allergy. Particular attention is paid to the potential of novel cellular tests such as basophil activation tests and component-resolved diagnosis with recombinant venom allergens in the diagnostic approach of patients with difficult diagnosis, i.e. cases in whom traditional venom specific IgE and skin tests yield equivocal or negative results. Finally, this review also covers the recent discoveries in the field of proteome research of Hymenoptera venoms and the selection of cell types for recombinant allergens production

IgE recognition of multiple novel Api m 10 isoforms evaluated by protein array technology

Background: Until now, two splice variants of the honeybee venom allergen Api m 10 have been described. Variant 2 exhibits IgE reactivity with approximately 50% of honeybee venom sensitized patients. Our proteomic analysis of honeybee venom gave an indication for the existence of additional Api m 10 isoforms. As such, further research was needed to identify these isoforms and investigate the impact of protein heterogeneity on IgE recognition. Method: Novel isoforms were searched by sequence analysis of cloned RT-PCR amplicons. All isoforms were produced as synthetic peptides or aglycosylated recombinant proteins and were spotted on nitrocellulose-coated glass slides. The colorimetric protein array technology was used to test IgE reactivity of the complete panel of isoforms using sera of 22 Api m 10 sensitized patients. Results: Nine additional Api m 10 isoforms were found, which derive from the same genomic locus by a complicated alternative splicing. Some truncated isoforms are produced by frameshifts which introduce an alternative stop codon. All 11 variants were obtained as synthetic peptides or purified bacterial recombinants. The colorimetric protein array showed differential IgE reactivity of different Api m 10 isoforms and allowed to predict an IgE epitope. Conclusion: Differential IgE reactivity of eleven Api m 10 isoforms was investigated using the colorimetric protein array technology. This approach shows some major benefits for testing IgE reactivity of a broad antigen panel. First, compared to many other technologies, only very low amounts of serum (25 µl) are needed. Second, in contrast to fluorescent arrays, the colorimetric signal detection allows the use of much cheaper scanners