Ellen L. Danneels

Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage.

Honey bee workers use venom for the defence of the colony and themselves when they are exposed to dangers and predators. It is produced by a long thin, convoluted, and bifurcated gland, and consists of several toxic proteins and peptides. The present study was undertaken in order to identify the mechanisms that protect the venom gland secretory cells against these harmful components. Samples of whole venom glands, including the interconnected reservoirs, were separated by two-dimensional gel electrophoresis and the most abundant protein spots were subjected to mass spectrometric identification using MALDI TOF/TOF-MS and LC MS/MS. This proteomic study revealed four antioxidant enzymes: CuZn superoxide dismutase (SOD1), glutathione-S-transferase sigma 1 isoform A (GSTS1), peroxiredoxin 2540 (PXR2540) and thioredoxin peroxidase 1 isoform A (TPX1). Although glutathione-S-transferase (GST) has also been associated with xenobiotic detoxification, the protein we found belongs to the GST Sigma class which is known to protect against oxidative stress only. Moreover, we could demonstrate that the GST and SOD activity of the venom gland was low and moderate, respectively, when compared to other tissues from the adult honey bee. Several proteins involved in other forms of stress were likewise found but it remains uncertain what their function is in the venom gland. In addition to major royal jelly protein 9 (MRJP9), already found in a previous proteomic study, we identified MRJP8 as second member of the MRJP protein family to be associated with the venom gland. Transcripts of both MRJPs were amplified and sequenced. Two endocuticular structural proteins were abundantly present in the 2D-gel and most probably represent a structural component of the epicuticular lining that protects the secretory cells from the toxins they produce.

Venom Proteins of the Parasitoid Wasp Nasonia vitripennis: Recent Discovery of an Untapped Pharmacopee

Adult females of Nasonia vitripennis inject a venomous mixture into its host flies prior to oviposition. Recently, the entire genome of this ectoparasitoid wasp was sequenced, enabling the identification of 79 venom proteins. The next challenge will be to unravel their specific functions, but based on homolog studies, some predictions already can be made. Parasitization has an enormous impact on hosts physiology of which five major effects are discussed in this review: the impact on immune responses, induction of developmental arrest, increases in lipid levels, apoptosis and nutrient releases. The value of deciphering this venom is also discussed.

Genome‐wide analysis of alternative reproductive phenotypes in honeybee workers

A defining feature of social insects is the reproductive division of labour, in which workers usually forego all reproduction to help their mother queen to reproduce. However, little is known about the molecular basis of this spectacular form of altruism. Here, we compared gene expression patterns between nonreproductive, altruistic workers and reproductive, non‐altruistic workers in queenless honeybee colonies using a whole‐genome microarray analysis. Our results demonstrate massive differences in gene expression patterns between these two sets of workers, with a total of 1292 genes being differentially expressed. In nonreproductive workers, genes associated with energy metabolism and respiration, flight and foraging behaviour, detection of visible light, flight and heart muscle contraction and synaptic transmission were overexpressed relative to reproductive workers. This implies they probably had a higher whole‐body energy metabolism and activity rate and were most likely actively foraging, whereas same‐aged reproductive workers were not. This pattern is predicted from evolutionary theory, given that reproductive workers should be less willing to compromise their reproductive futures by carrying out high‐risk tasks such as foraging or other energetically expensive tasks. By contrast, reproductive workers mainly overexpressed oogenesis‐related genes compared to nonreproductive ones. With respect to key switches for ovary activation, several genes involved in steroid biosynthesis were upregulated in reproductive workers, as well as genes known to respond to queen and brood pheromones, genes involved in TOR and insulin signalling pathways and genes located within quantitative trait loci associated with reproductive capacity in honeybees. Overall, our results provide unique insight into the molecular mechanisms underlying alternative reproductive phenotypes in honeybee workers.

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.

Early changes in the pupal transcriptome of the flesh fly Sarcophagha crassipalpis to parasitization by the ectoparasitic wasp, Nasonia vitripennis

We investigated changes in the pupal transcriptome of the flesh fly Sarcophaga crassipalpis, 3 and 25 h after parasitization by the ectoparasitoid wasp, Nasonia vitripennis. These time points are prior to hatching of the wasp eggs, thus the results document host responses to venom injection, rather than feeding by the wasp larvae. Only a single gene appeared to be differentially expressed 3 h after parasitization. However, by 25 h, 128 genes were differentially expressed and expression patterns of a subsample of these genes were verified using RT-qPCR. Among the responsive genes were clusters of genes that altered the flys metabolism, development, induced immune responses, elicited detoxification responses, and promoted programmed cell death. Envenomation thus clearly alters the metabolic landscape and developmental fate of the fly host prior to subsequent penetration of the pupal cuticle by the wasp larva. Overall, this study provides new insights into the specific action of ectoparasitoid venoms.

Preliminary analysis of loss rates of honey bee colonies during winter 2015/16 from the COLOSS survey

In this short note we present comparable loss rates of honey bee colonies during winter 2015/16 from 29 countries, obtained with the COLOSS questionnaire. Altogether, we received valid answers from 19,952 beekeepers. These beekeepers collectively wintered 421,238 colonies, and reported 18,587 colonies with unsolvable queen problems and 32,048 dead colonies after winter. This gives an overall loss rate of 12.0% (95% confidence interval 11.8–12.2%) during winter 2015/16, with marked differences among countries. Beekeepers in the present study assessed 7.6% (95% CI 7.4–7.8%) of their colonies as dead or empty, and 4.4% (95% CI 4.3–4.5%) as having unsolvable queen problems after winter. The overall analysis showed that small operations suffered higher losses than larger ones. A table with detailed results and a map showing response and relative risks at regional level are presented.

How the venom from the ectoparasitoid wasp Nasonia vitripennis exhibits anti-inflammatory properties on mammalian cell lines

With more than 150,000 species, parasitoids are a large group of hymenopteran insects that inject venom into and then lay their eggs in or on other insects, eventually killing the hosts. Their venoms have evolved into different mechanisms for manipulating host immunity, physiology and behavior in such a way that enhance development of the parasitoid young. The venom from the ectoparasitoid Nasonia vitripennis inhibits the immune system in its host organism in order to protect their offspring from elimination. Since the major innate immune pathways in insects, the Toll and Imd pathways, are homologous to the NF-κB pathway in mammals, we were interested in whether a similar immune suppression seen in insects could be elicited in a mammalian cell system. A well characterized NF-κB reporter gene assay in fibrosarcoma cells showed a dose-dependent inhibition of NF-κB signaling caused by the venom. In line with this NF-κB inhibitory action, N. vitripennis venom dampened the expression of IL-6, a prototypical proinflammatory cytokine, from LPS-treated macrophages. The venom also inhibited the expression of two NF-κB target genes, IκBα and A20, that act in a negative feedback loop to prevent excessive NF-κB activity. Surprisingly, we did not detect any effect of the venom on the early events in the canonical NF-κB activation pathway, leading to NF-κB nuclear translocation, which was unaltered in venom-treated cells. The MAP kinases ERK, p38 and JNK are other crucial regulators of immune responses. We observed that venom treatment did not affect p38 and ERK activation, but induced a prolonged JNK activation. In summary, our data indicate that venom from N. vitripennis inhibits NF-κB signaling in mammalian cells. We identify venom-induced up regulation of the glucocorticoid receptor-regulated GILZ as a most likely molecular mediator for this inhibition.

Proteomics of the Venom of the Parasitoid Nasonia vitripennis

Publisher Summary This chapter discusses the proteomics of the venom of the parasitoid nasonia virtipennis. Nasonia vitripennis is an ectoparasitoid that uses host flies as a food source for its progeny, whereby the venom of adult females is used to subdue the host. The venom of this tiny wasp is able to elicit several host responses ranging from an altered immunity to developmental changes. Over the last few years, the proteomic research of venomous substances has gained more and more interest and several powerful tools have become available. Animal venoms contain a complex mixture of proteins, enzymes, peptides, and small organic compounds. Venomics, the analysis of venom proteomes, has gained a lot of interest because venoms are used for predation or defense and contain toxic compounds that are associated with specific pathologies. Identification of venom components in parasitoid wasps was restricted due to the technological limitations and lack of genome sequences.

Exploring the Potential of Venom from Nasonia vitripennis as Therapeutic Agent with High-Throughput Screening Tools.

The venom from the ectoparasitoid wasp Nasonia vitripennis (Hymenoptera: Pteromalidae) contains at least 80 different proteins and possibly even more peptides or other small chemical compounds, demonstrating its appealing therapeutic application. To better understand the dynamics of the venom in mammalian cells, two high-throughput screening tools were performed. The venom induced pathways related to an early stress response and activated reporters that suggest the involvement of steroids. Whether these steroids reside from the venom itself or show an induced release/production caused by the venom, still remains unsolved. The proinflammatory cytokine IL-1β was found to be down-regulated after venom and LPS co-treatment, confirming the anti-inflammatory action of N. vitripennis venom. When analyzing the expression levels of the NF-κB target genes, potentially not only the canonical but also the alternative NF-κB pathway can be affected, possibly explaining some counterintuitive results. It is proposed that next to an NF-κB binding site, the promoter of the genes tested by the PCR array may also contain binding sites for other transcription factors, resulting in a complex puzzle to connect the induced target gene with its respective transcription factor. Interestingly, Nasonia venom altered the expression of some drug targets, presenting the venom with an exciting therapeutical potential.

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.