Matthias Van Vaerenbergh

Finding the missing honey bee genes: Lessons learned from a genome upgrade

BackgroundThe first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes.ResultsHere, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data.ConclusionsLessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

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.

Exploring the hidden honeybee (Apis mellifera) venom proteome by integrating a combinatorial peptide ligand library approach with FTMS.

UNLABELLED At present, 30 compounds have been described in the venom of the honeybee, and 16 of them were confirmed by mass spectrometry. Previous studies typically combined 2-D PAGE with MALDI-TOF/TOF MS, a technology which now appears to lack sensitivity to detect additional venom compounds. Here, we report an in-depth study of the honeybee venom proteome using a combinatorial peptide ligand library sample pretreatment to enrich for minor components followed by shotgun LC-FT-ICR MS analysis. This strategy revealed an unexpectedly rich venom composition: in total 102 proteins and peptides were found, with 83 of them never described in bee venom samples before. Based on their predicted function and subcellular location, the proteins could be divided into two groups. A group of 33 putative toxins is proposed to contribute to venom activity by exerting toxic functions or by playing a role in social immunity. The other group, considered as venom trace molecules, appears to be secreted for their functions in the extracellular space, or is unintentionally secreted by the venom gland cells due to insufficient protein recycling or co-secretion with other compounds. In conclusion, our approach allowed to explore the hidden honeybee venom proteome and extended the list of potential venom allergens. BIOLOGICAL SIGNIFICANCE This study dug deeper into the complex honeybee venom proteome than ever before by applying a highly performing sample pretreatment and mass spectrometric technology. We present putative biological functions for all identified compounds, largely extending our knowledge of the venom toxicity. In addition, this study offers a long list of potential new venom allergens.

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 chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4.

Unraveling the venom proteome of the bumblebee (Bombus terrestris) by integrating a combinatorial peptide ligand library approach with FT-ICR MS

Within the Apidae, the largest family of bees with over 5600 described species, the honeybee is the sole species with a well studied venom proteome. So far, only little research has focused on bumblebee venom. Recently, the genome sequence of the European large earth bumblebee (Bombus terrestris) became available and this allowed the first in-depth proteomic analysis of its venom composition. We identified 57 compounds, with 52 of them never described in bumblebee venom. Remarkably, 72% of the detected compounds were found to have a honeybee venom homolog, which reflects the similar defensive function of both venoms and the high degree of homology between both genomes. However, both venoms contain a selection of species-specific toxins, revealing distinct damaging effects that may have evolved in response to species-specific attackers. Further, this study extends the list of potential venom allergens. The availability of both the honeybee and bumblebee venom proteome may help to develop a strategy that solves the current issue of false double sensitivity in allergy diagnosis, which is caused by cross-reactivity between both venoms. A correct diagnosis is important as it is recommended to perform an immunotherapy with venom of the culprit species.

Hymenoptera Venoms: Toxicity, Components, Stability, and Standardization

Bees, wasps, and ants possess predatory and/or defensive venoms that contain many toxic compounds. Diverse life cycles of this enormous group of insects implicate a large variability in venom compositions. Several venom compounds have been reported to cause an allergic reaction in humans, suggesting that a good knowledge of the composition of venoms and the structure of allergens is a prerequisite for the accurate diagnosis and treatment of insect venom allergy. A large group of proteins and peptides that is even more complex due to protein heterogeneity and posttranslational modifications represents a huge source of structurally diverse and biologically active toxins with high potency and selectivity for a wide range of targets. Due to the presence of similar protein allergens in multiple Hymenoptera venoms, cross-reactivity occurs between venoms from different species. Concerning the treatment of allergy caused by stinging insects, obtaining highly standardized allergens is crucial in allergy diagnosis.