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Introduction: Changes in the cytokine profile from type 2 to type 1 together with the induction of regulatory cells are expected during hymenoptera venom immunotherapy (VIT). The present study was aimed to investigate the changes in type 1, type 2, and regulatory cytokines induced by a Vespula spp. VIT in patients with anaphylaxis to Vespa velutina., Methods: Twenty consecutive patients with anaphylaxis due to Vespa velutina were treated with Vespula spp. VIT. Serum cytokines (IL-4, IL-5, IL-10, IL-13, and IFN-ɣ) were measured at baseline, 6, and 12 months after starting VIT., Results: A significant increase in serum IFN-y was detected after 6 and 12 months of VIT. An increase in serum IL-10 and a decrease in IL-5 were observed after 12 months. IL-4 was undetectable all along the study, and an unexpected increase of IL-13 was present at 12 months of treatment., Conclusion: Vespula spp. VIT seems to be able to induce a shift to type 1 cytokine production measured through IFN-y levels and IL-10 production after, at least, 6 and 12 months of VIT, respectively., (© 2024 S. Karger AG, Basel.)

Wasp allergy with a diagnostic profile of double sensitizations to vespid venom is a frequent clinical problem in areas where different genera of wasps are present. Identification of the insect responsible for serious reactions poses a diagnostic challenge as the only effective treatment to date is immunotherapy based on the specific venom. In southern Europe, the double sensitization to Vespula and Polistes venoms is highly frequent. It has been shown that the major allergenic proteins (Phospholipase A1 and Antigen 5) share sequences across the different genera and species, which would be the cause of cross-reactivity. Additionally, the minor allergens (Dipeptidyl-peptidases, Vitellogenins) have been found to share partial sequence identity. Furthermore, venom contains other homologous proteins whose allergenic nature still remains to be clarified. The traditional diagnostic tools available are insufficient to discriminate between allergy to Vespula and Polistes in a high number of cases. IgE inhibition is the technique that best identifies the cross-reactivity. When a double sensitization has indeed been shown to exist or great uncertainty surrounds the primary sensitization, therapy with two venoms is advisable to guarantee the safety of the patient. In this case, a strategy involving alternate administration that combines effectiveness with efficiency is possible.

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila-wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps' side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

Scavenger receptors (SRs) are a family of pattern recognition receptors (PRRs) in the immune system. They are required for phagocytosis and act as co-receptors of Toll-like receptors to regulate immune signaling pathways in the fight against pathogens. Little is known about the function of SRs in insects. Here, we reported on a member of the SR family from the parasitic wasp Micropilits mediator (designated MmSR-B1) that is responsive to bacterial infection. The recombinant extracellular CD36 domain of MmSR-B1 produced in Escherichia coli cells is capable of binding to peptidoglycans and bacterial cells, causing agglutination of bacteria. Furthermore, we demonstrated that double-stranded RNA-mediated knockdown of MmSR-B1 impedes hemocyte phagocytosis and downregulates the expression of antimicrobial peptide (AMP) genes defensins and hymenoptaecins. Knockdown of MmSR-B1 led to increased death of the wasps when challenged by bacteria. Our study suggests that MmSR-B1 mediates phagocytosis and the production of AMPs in M. mediator wasps., (Copyright © 2021. Published by Elsevier Ltd.)

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts' hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp's immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite's simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity., Competing Interests: The authors have declared that no competing interests exist.

Many organisms are able to elicit behavioral change in other organisms. Examples include different microbes (e.g., viruses and fungi), parasites (e.g., hairworms and trematodes), and parasitoid wasps. In most cases, the mechanisms underlying host behavioral change remain relatively unclear. There is a growing body of literature linking alterations in immune signaling with neuron health, communication, and function; however, there is a paucity of data detailing the effects of altered neuroimmune signaling on insect neuron function and how glial cells may contribute toward neuron dysregulation. It is important to consider the potential impacts of altered neuroimmune communication on host behavior and reflect on its potential role as an important tool in the "neuro-engineer" toolkit. In this review, we examine what is known about the relationships between the insect immune and nervous systems. We highlight organisms that are able to influence insect behavior and discuss possible mechanisms of behavioral manipulation, including potentially dysregulated neuroimmune communication. We close by identifying opportunities for integrating research in insect innate immunity, glial cell physiology, and neurobiology in the investigation of behavioral manipulation.

Parasitoids are ubiquitous in natural ecosystems. Parasitic strategies are highly diverse among parasitoid species, yet their underlying genetic bases are poorly understood. Here, we focus on the divergent adaptation of a specialist and a generalist drosophilid parasitoids. We find that a novel protein (Lar) enables active immune suppression by lysing the host lymph glands, eventually leading to successful parasitism by the generalist. Meanwhile, another novel protein (Warm) contributes to a passive strategy by attaching the laid eggs to the gut and other organs of the host, leading to incomplete encapsulation and helping the specialist escape the host immune response. We find that these diverse parasitic strategies both originated from lateral gene transfer, followed with duplication and specialization, and that they might contribute to the shift in host ranges between parasitoids. Our results increase our understanding of how novel gene functions originate and how they contribute to host adaptation.

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. We find that Drosophila employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. We show that neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming., Competing Interests: SM, MS, AK, MG, MJ, PU, KG, JS, TM No competing interests declared, (© 2020, Madhwal et al.)

Paper wasps (Polistes dominula), parasitized by the strepsipteran Xenos vesparum, are castrated and desert the colony to gather on plants where the parasite mates and releases primary larvae, thus completing its lifecycle. One of these plants is the trumpet creeper Campsis radicans: in a previous study the majority of all wasps collected from this plant were parasitized and focused their foraging activity on C. radicans buds. The unexpected prevalence and unusual feeding strategy prompted us to investigate the influence of this plant on wasp behavior and physiology through a multidisciplinary approach. First, in a series of laboratory bioassays, we observed that parasitized wasps spent more time than non-parasitized ones on fresh C. radicans buds, rich of extra-floral nectaries (EFNs), while the same wasps ignored treated buds that lacked nectar drops. Then, we described the structure and ultra-structure of EFNs secreting cells, compatible with the synthesis of phenolic compounds. Subsequently, we analysed extracts from different bud tissues by HPLC-DAD-MS and found that verbascoside was the most abundant bioactive molecule in those tissues rich in EFNs. Finally, we tested the immune-stimulant properties of verbascoside, as the biochemical nature of this compound indicates it might function as an antibacterial and antioxidant. We measured bacterial clearance in wasps, as a proxy for overall immune competence, and observed that it was enhanced after administration of verbascoside-even more so if the wasp was parasitized. We hypothesize that the parasite manipulates wasp behavior to preferentially feed on C. radicans EFNs, since the bioactive properties of verbascoside likely increase host survival and thus the parasite own fitness., Competing Interests: The authors have declared that no competing interests exist.

Background: Hymenoptera venom allergy (HVA) is of great concern because of the possibility of anaphylaxis, which may be fatal. Venom immunotherapy (VIT) is the only disease-modifying treatment in HVA and, although efficient, its mechanism remains partially unknown. Gene expression analysis may be helpful for establishing a proper model of tolerance induction during the build-up phase of VIT. The present study aimed to analyze how the start of VIT changes the expression of 15 selected genes., Methods: Forty-five patients starting VIT with a wasp venom allergy were enrolled. The diagnosis was established based on anaphylaxis history (third or fourth grade on the Mueller scale) and positive soluble immunoglobulin E and/or skin tests. Two blood collections were performed in the patient group: before and after 3 months of VIT. One sample was taken in the control group. Gene expression analysis was performed using a reverse transcriptase-polymerase chain reaction with microfluidic cards and normalized to the 18S housekeeping gene., Results: Commd8 was the only gene that changed expression significantly after the start of VIT (p = 0.012). Its expression decreased towards the levels observed in the healthy controls. Twelve out of 15 genes (commd8, cldn1, cngb3, fads1, hes6, hla-drb5, htr3b, prlr, slc16a4, snx33, socs3 and twist2) revealed a significantly different expression compared to the healthy controls., Conclusions: The present study shows that commd8 changes significantly its expression during initial phase of VIT. This gene might be a candidate for VIT biomarker in future studies., (© 2020 John Wiley & Sons, Ltd.)

Female parasitoid wasps normally inject virulence factors together with eggs into their host to counter host immunity defenses. A newly identified RhoGAP protein in the venom of Microplitis mediator compromises the cellular immunity of its host, Helicoverpa armigera. RhoGAP1 proteins entered H. armigera hemocytes, and the host cellular cytoskeleton was disrupted. Depletion of MmGAP1 by injection of dsRNA or antibody increased the wasp egg encapsulation rate. An immunoprecipitation assay of overexpressed MmGAP1 protein in a Helicoverpa cell line showed that MmGAP1 interacts with many cellular cytoskeleton associated proteins as well as Rho GTPases. A yeast two-hybrid and a pull-down assay demonstrated that MmGAP1 interacts with H. armigera RhoA and Cdc42. These results show that the RhoGAP protein in M. mediator can destroy the H. armigera hemocyte cellular cytoskeleton, restrain host cellular immune defense, and increase the probability of successful parasitism., Competing Interests: Declaration of competing interest The authors have no financial conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts., Results: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes., Conclusions: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.

Body size reduction, also known as miniaturization, is an important evolutionary process that affects a number of physiological and phenotypic traits and helps animals conquer new ecological niches. However, this process is poorly understood at the molecular level. Here, we report genomic and transcriptomic features of arguably the smallest known insect-the parasitoid wasp, Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae). In contrast to expectations, we find that the genome and transcriptome sizes of this parasitoid wasp are comparable to other members of the Chalcidoidea superfamily. Moreover, compared to other chalcid wasps the gene content of M. amalphitanum is remarkably conserved. Intriguingly, we observed significant changes in M. amalphitanum transposable element dynamics over time, in which an initial burst was followed by suppression of activity, possibly due to a recent reinforcement of the genome defense machinery. Overall, while the M. amalphitanum genomic data reveal certain features that may be linked to the unusual biological properties of this organism, miniaturization is not associated with a large decrease in genome complexity., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs are small non-coding molecules playing a significant regulatory role in several allergic diseases. However their role in tolerance induction remains unclear. The aim of this study was to determine the expression of selected microRNAs during the first three months of wasp venom immunotherapy (VIT). 5 adult patients with a history of severe systemic reactions after stinging by wasps and confirmed sensitization were included. Venous blood samples were collected before VIT, 24 hours after completing its initial phase and after 3 months of the maintenance therapy. A control group was comprised of 5 healthy individuals with no history of allergy. In the blood samples expression of 96 microRNAs was determined with the use of microfluidic cards. In a statistical analysis the expression was compared between the study groups as well as between the pre- and post-VIT samples. Significant differences were found between the patients with wasp venom allergy and the healthy controls in the expression of miR-601 and miR-1201 upregulated in allergic patients at every time point ( p = 0.04; p = 0.015, respectively). During VIT profile of microRNA was changing with lower expression of 6 microRNAs (including miR-182, miR-342, miR-375) and higher of 11 microRNAs (including let-7d, miR-34b, miR-143). To conclude, VIT has led to some changes in the expression of microRNA associated with Th2-type inflammation and tolerance induction.

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster's immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution., Competing Interests: The authors have declared that no competing interests exist.

Biological control using entomopathogens and natural enemies is an ecofriendly method for pest management in agriculture. Biological control agents often can be simultaneously employed and compatibility between agents may improve pest suppression. We investigated the influence of the entomopathogen Bacillus thuringiensis (Bt) on the immune system of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) to determine if such changes impact parasitization by Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). The immune response of surviving D. saccharalis larvae fed with an LC 25 dosage of a Bt-based biopesticide (Dipel®) was analyzed (total hemocyte count, hemocyte adhesion, and activities of phenoloxidase and lysozyme). Furthermore, the suitability of surviving Bt-fed larvae as hosts for C. flavipes was assessed by measuring parasitoid attributes such as number and size of teratocytes, weight of pupae, length of adult female tibia and number of emerged adults. Total hemocyte count, but not hemocyte adhesion, total protein content and phenoloxidase activity increased in the hemolymph of non-parasitized Bt-fed larvae (Bt-NP) compared to control larvae (NBt-NP). Lysozyme activity increased only after parasitization without Bt exposure (NBt-P). After parasitization, the immunological parameters activated in Bt-NP larvae decreased to levels at or below those observed in control larvae, showing that C. flavipes can regulate the activated immune response of Bt-fed larvae. The development of C. flavipes was not impaired in Bt-fed larval hosts (Bt-P); no changes were observed for teratocyte size, weight of pupal mass, length of hind tibia and number of adults emerged., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Plants emit a specific blend of volatiles in response to herbivory and these volatiles, which often attract predators and parasitoids function as an indirect plant defense. The impact of plant volatiles in shaping herbivore defenses is unclear. Here, we report that specific plant volatiles induce immune responses in the polyphagous herbivore, Spodoptera litura. We characterized the hemocyte profile and established their functional significance with respect to ontogeny and exposure to specific plant volatiles. Fifth instar larvae showed the highest number and hemocytes diversity. We characterized seven different types of hemocytes, of which granulocytes performed phagocytosis, oenocytoids showed melanization activity, and plasmatocytes along with granulocytes and oenocytoids were found to be involved in encapsulation. Among the six volatiles tested, exposure to (E)-β-ocimene caused the highest increase in total hemocytes number (THC) followed by linalool and (Z)-3-hexenyl acetate exposure. Although THC did not differ between these three volatile treatments, circulating hemocytes diversity varied significantly. (E)-β-ocimene exposure showed higher number of plasmatocytes and phenol oxidase activity. The interaction of the parasitic wasp Bracon brevicornis with (E)-β-ocimene exposed larvae was poor in terms of delayed paralysis and lower egg deposition. In choice assays, the wasp showed clear preference towards control larvae indicating (E)-β-ocimene treatment renders the host unattractive. Hemocyte profiles post-parasitoid exposure and (E)-β-ocimene treatment were similar indicating cue-based priming. When challenged with Bacillus thuringiensis, linalool exposure resulted in the highest survival as compared to other volatiles. Our results show that specific HIPVs can modulate cellular immunity of S. litura, revealing a new role for HIPVs in tri-trophic interactions.

Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to protect their offspring from the host immune response. The hymenopteran species of the genus Leptopilina , parasites of Drosophila , rely on the injection of a venom which contains proteins and peculiar vesicles (hereafter venosomes). We show here that the injection of purified L. boulardi venosomes is sufficient to impair the function of the Drosophila melanogaster lamellocytes, a hemocyte type specialized in the defense against wasp eggs, and thus the parasitic success of the wasp. These venosomes seem to have a unique extracellular biogenesis in the wasp venom apparatus where they acquire specific secreted proteins/virulence factors and act as a transport system to deliver these compounds into host lamellocytes. The level of venosomes entry into lamellocytes of different Drosophila species was correlated with the rate of parasitism success of the wasp, suggesting that this venosome-cell interaction may represent a new evolutionary level of host-parasitoid specificity.

Eukaryotic initiation factor 4E (eIF4E) is regulated during the innate immune response. However, its translational regulation under innate immune suppression remains largely unexplored. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus harbored by the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we generated eIF4E dsRNA and used it to silence the eIF4E gene of S. litura, resulting in a hallmark immunosuppressive phenotype characterized by increased apoptosis of hemocytes and retardation of head capsule width development. In response to natural parasitism, loss of eIF4E function was associated with similar immunosuppression, and we detected no significant differences between the response to parasitism and treatment with eIF4E RNAi. Under MbBV infection, eIF4E overexpression significantly suppressed MbBV-induced increase in apoptosis and suppressed apoptosis to the same extent as co-expression of both eIF4E and eIF4A. There were no significant differences between MbBV-infected and uninfected larvae in which eIF4E was overexpressed. More importantly, in the eIF4E RNAi strain, eIF4A RNAi did not increase apoptosis. Collectively, our results indicate that eIF4E plays a nodal role in the MbBV-suppressed innate immune response via the eIF4E-eIF4A axis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies., Competing Interests: The authors have declared that no competing interests exist.

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism., Competing Interests: The authors have declared that no competing interests exist.

Background: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity., Objectives: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE., Methods: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes., Results: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2., Conclusion: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Wasp venom is a potentially important natural drug, but it can cause hypersensitivity reactions. The purpose of the present study was to systematically study the epitopes of wasp venom., Methods: Using a random 12-peptide phage library, we performed antibody-binding epitope panning on ten serum samples from wasp sting victims at 3 h and 4 days after the sting. The panning epitopes were identified by high-throughput sequencing and matched with wasp venom proteins by BLAST. The panned antibody-binding epitopes were verified by ELISA., Results: A total of 35 specific potential wasp venom epitopes in 4 days were identified. Amongst them, twelve peptide epitopes were matched with nine wasp venom proteins, namely, vitellogenin precursor, hexamerin 70b precursor, venom carboxylesterase-6 precursor, MRJP5, major royal jelly protein 8 precursor, venom acid phosphatase Acph-1 precursor, phospholipase A2, venom serine protease 34 precursor, and major royal jelly protein 9 precursor. The changes in serum IgM antibodies induced by wasp venom were confirmed by ELISA based on the 12 peptide epitopes., Conclusion: The nine wasp venom proteins are potential allergens, which should be excluded or modified in the potential biomedical applications of wasp venom., (© 2018 The Author(s).)

It has been recently suggested that female mate choice, based on sexually selected ornaments, is an important component of social wasps' reproductive biology. The correlates of male ornaments that could be of a female's interest, however, remain to be investigated. Males of the Neotropical paper wasp Polistes simillimus have sexually dimorphic melanin-based black spots on their faces. In this species, male spots work like sexual ornaments, as it has been experimentally demonstrated that females prefer sexual partners with a higher proportion of black pigment on their faces. We have shown that, under laboratory conditions, male sexual ornamentation positively predicts the strength of the melanization immune response and longevity. Therefore, in P. simillimus, melanin-based facial patterns (ornaments) seem to be honest indicators of male quality., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acute inflammation can cause serious tissue damage and disease in physiologically-challenged organisms. The precise mechanisms leading to these detrimental effects remain to be determined. In this study, we utilize a reproducible means to induce cellular immune activity in Drosophila larvae in response to mechanical stress. That is, forceps squeeze-administered stress induces lamellocytes, a defensive hemocyte type that normally appears in response to wasp infestation of larvae. The posterior signaling center (PSC) is a cellular microenvironment in the larval hematopoietic lymph gland that is vital for lamellocyte induction upon parasitoid attack. However, we found the PSC was not required for mechanical stress-induced lamellocyte production. In addition, we observed that mechanical injury caused a systemic expression of Unpaired3. This cytokine is both necessary and sufficient to activate the cellular immune response to the imposed stress. These findings provide new insights into the communication between injured tissues and immune system induction, using stress-challenged Drosophila larvae as a tractable model system., (Copyright © 2018. Published by Elsevier Inc.)

Background: Venom immunotherapy is effective in preventing systemic allergic reactions (SARs), but the diagnosis of venom allergy is problematic., Objective: To compare the performance of component-resolved diagnosis and conventional tests in patients referred for venom immunotherapy., Methods: We measured serum-specific immunoglobulin E to yellowjacket and honeybee venoms (Ves v 1 and Ves v 5 and Api m 1), cross-reactive carbohydrate determinants, serum basal tryptase (ImmunoCAP, ThermoFisher Scientific, Uppsala, Sweden), and skin prick test reactions in 84 patients referred to receive venom immunotherapy. History of SAR and its severity were evaluated., Results: Of the 78 patients with suspected yellowjacket venom (YJV) allergy, a history of SAR was confirmed in 47 (60%) and 31 (40%) had a non-SAR reaction. The most accurate tests to confirm venom allergy after a SAR were serum-specific immunoglobulin E to yellowjacket whole-venom extract spiked with Ves v 5 (area under the curve 0.87, 95% confidence interval 0.77-0.97, P < .001) and Ves v 5 (area under the curve 0.86, 95% confidence interval 0.76-0.96, P < .001). Sensitization to Ves v 1 was infrequent and its area under the curve was low (0.62, 95% confidence interval 0.47-0.76, P = .106). Sensitivity of the YJV skin prick test was 86%, but its specificity was low at 54%. Double sensitization to yellowjacket and honeybee occurred frequently in skin prick tests. Of the patients without a SAR, 26% showed a positive reaction to YJV in any serum test and 46% showed a positive reaction in skin tests., Conclusion: Specific immunoglobulin E to the YJV spiked with Ves v 5 confirmed the allergy after a SAR. A history of SAR should be confirmed before testing, because venom sensitization is frequent in other types of reactions., (Copyright © 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The long-term protection provided by venom immunotherapy (VIT) is related to the dose administered and to its long duration; the latter, however, becomes inconvenient for patients in countries like Greece, with many islanders or inhabitants of distant mountainous areas. Maintenance interval prolongation reduces the number of office visits - saving time and money - and as a consequence contributes to the patients' compliance. The aim of this prospective study was to evaluate the safety and efficacy of VIT on a progressively prolonged maintenance interval (PPMI)., Method: 450 venom-allergic patients were reviewed for participation in our study; all of them were initially treated with a modified rush or an ultrarush protocol using freshly reconstituted, pure venoms. Upon reaching the maintenance dose, the VIT interval was scheduled to be gradually prolonged - by 1 week each time - aiming at a maximal interval of 26 weeks., Results: 267/450 patients consented to participate in our VIT PPMI protocol: 98 were treated with vespid(s) venom, 142 with honeybee venom, and 27 with both. The mean duration of patient follow-up was 9.1 ± 4.2 years. The majority of systemic reactions due to VIT injections occurred up to the 8-weeks PPMI; few additional reactions were documented in a small fraction (2.9%) of our patient population beyond 9 weeks and up to 16 weeks; all were caused by honeybee VIT. No reactions were observed during VIT administration at the 26-week interval. Ninety-six patients reported 204 field sting occurrences by the culprit insect. Ten systemic reactions (8 mild and 2 moderate in severity) were registered between the 9- and 18-week PPMI; the honeybee was the culprit insect in all cases. 108 field stings by the offending insect were sustained beyond the 20- and up to the 26-week PPMI; there were no reactions at all., Conclusions: Progressively prolonging the VIT maintenance interval up to 26 weeks appears to be safe and efficacious., (© 2018 S. Karger AG, Basel.)

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cross-reactions between Polistes dominula and Vespula species are common in southern Europe. Currently, only CAP-inhibition demonstrates high accuracy in identifying genuine sensitizations, but this method is time-consuming and expensive, so a new approach is required. This study investigates skin tests, molecular diagnostics, total IgE (tIgE), and the Ves v 5/Pol d 5 (or vice versa) ratio. The ratio generated low-accuracy results and poor agreement with CAP-inhibition, and we did not find any agreement between CAP-inhibition test and double values of Ves v 5/Pol d 5. Nevertheless, a slight diagnostic improvement was obtained when Ves v 5/tIgE and Pol d 5/tIgE were measured., (© 2018 S. Karger AG, Basel.)

Hematopoietic stem/progenitor cells in the adult mammalian bone marrow ensure blood cell renewal. Their cellular microenvironment, called 'niche', regulates hematopoiesis both under homeostatic and immune stress conditions. In the Drosophila hematopoietic organ, the lymph gland, the posterior signaling center (PSC) acts as a niche to regulate the hematopoietic response to immune stress such as wasp parasitism. This response relies on the differentiation of lamellocytes, a cryptic cell type, dedicated to pathogen encapsulation and killing. Here, we establish that Toll/NF-κB pathway activation in the PSC in response to wasp parasitism non-cell autonomously induces the lymph gland immune response. Our data further establish a regulatory network where co-activation of Toll/NF-κB and EGFR signaling by ROS levels in the PSC/niche controls lymph gland hematopoiesis under parasitism. Whether a similar regulatory network operates in mammals to control emergency hematopoiesis is an open question.

A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species.

The evolutionary success of parasitoid wasps, a highly diverse group of insects widely used in biocontrol, depends on a variety of life history strategies in conflict with those of their hosts [1]. Drosophila melanogaster is a natural host of parasitic wasps of the genus Leptopilina. Attack by L. boulardi (Lb), a specialist wasp to flies of the melanogaster group, activates NF-κB-mediated humoral and cellular immunity. Inflammatory blood cells mobilize and encapsulate Lb eggs and embryos [2-5]. L. heterotoma (Lh), a generalist wasp, kills larval blood cells and actively suppresses immune responses. Spiked virus-like particles (VLPs) in wasp venom have clearly been linked to wasps' successful parasitism of Drosophila [6], but the composition of VLPs and their biotic nature have remained mysterious. Our proteomics studies reveal that VLPs lack viral coat proteins but possess a pharmacopoeia of (1) the eukaryotic vesicular transport system, (2) immunity, and (3) previously unknown proteins. These novel proteins distinguish Lh from Lb VLPs; notably, some proteins specific to Lh VLPs possess sequence similarities with bacterial secretion system proteins. Structure-informed analyses of an abundant Lh VLP surface and spike-tip protein, p40, reveal similarities to the needle-tip invasin proteins SipD and IpaD of Gram-negative bacterial type-3 secretion systems that breach immune barriers and deliver virulence factors into mammalian cells. Our studies suggest that Lh VLPs represent a new class of extracellular organelles and share pathways for protein delivery with both eukaryotic microvesicles and bacterial surface secretion systems. Given their mixed prokaryotic and eukaryotic properties, we propose the term mixed-strategy extracellular vesicle (MSEV) to replace VLP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Venom immunotherapy (VIT) is highly effective in preventing allergic reactions to insect stings, but the appropriate venom must be used to achieve clinical protection. In patients with multiple positive results to venoms, molecular allergy diagnostics or CAP-inhibition may identify the causative venom. Concerning allergy to venom from Polistes spp. it has been proposed that only the European species P. dominulus should be used for VIT. However, this recommendation is not present in any international guideline. Using both laboratory and clinical data, we aimed to evaluate the reliability of this proposal., Methods: We performed an in vitro study using CAP-inhibition to determine sensitization of 19 patients allergic to Polistes venom. The clinical study included 191 patients with positive tests to Polistes treated with VIT, 102 were treated with P. dominulus and 89 were treated with a mix of American Polistes (mAP)., Results: The difference in % of inhibition was significant concerning inhibition of P. dominulus sIgE by P. dominulus venom (79.8%) compared with inhibition by mAP venom (64.2%) and not significant concerning the inhibition of mAP sIgE by P. dominulus venom (80.1%) and by mAP venom (73.6%). Instead, the clinical protection from stings was not statistically different between the two kinds of venom., Conclusion: The data from CAP inhibition would suggest that the choice of either P. dominulus venom or mAP venom for VIT is appropriate in patients with CAP inhibition higher than 70%, but the clinical data show the same odds of protection from stings using for VIT P. dominulus or mAP venom.

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune receptors that specifically recognize peptidoglycans (PGNs) on the surface of a number of pathogens. Here, we have identified and characterized six PGRPs from endoparasitoid wasp, Microplitis mediator (MmePGRPs). To understand the roles of PGRPs in parasitoid wasps, we analyzed their evolutionary relationship and orthology, expression profiles during different developmental stages, and transcriptional expression following infection with Gram-positive and -negative bacteria and a fungus. MmePGRP-S1 was significantly induced in response to pathogenic infection. This prompted us to evaluate the effects of RNA interference mediated gene specific knockdown of MmePGRP-S1. The knockdown of MmePGRP-S1 (iMmePGRP-S1) dramatically affected wasps' survival following challenge by Micrococcus luteus, indicating the involvement of this particular PGRP in immune responses against Gram-positive bacteria. This action is likely to be mediated by the Toll pathway, but the mechanism remains to be determined. MmePGRP-S1 does not play a significant role in anti-fungal immunity as indicated by the survival rate of iMmePGRP-S1 wasps. This study provides a comprehensive characterization of PGRPs in the economically important hymenopteran species M. mediator., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

Background: Skin test reactivity to hymenoptera venom and venom-specific IgE are important for diagnosing venom allergy and deciding on the appropriate allergen for venom immunotherapy (VIT). Longitudinal data on skin test reactivity during VIT and their correlation with venom-specific immunoglobulin (Ig)E and IgG are scarce., Methods: We retrospectively analyzed shifts in skin test reactivity and serum levels of venom-specific IgE and IgG in patients allergic to hymenoptera venom before the initiation of VIT with ultrarush therapy and after ≥3 years of VIT., Results: Fifty-four patients received ultrarush desensitization and subsequent VIT with wasp venom, 26 with honeybee venom, and 8 with both wasp and honeybee venom. Hymenoptera-specific skin test reactivity decreased during VIT in most patients, and became negative in 8% of the wasp-allergic patients and in 25% of the honeybee-allergic patients. Serum levels of venom-specific IgE positively correlated to skin test reactivity before VIT, but did not change significantly during VIT. IgG serum levels and the IgG/IgE ratio increased during VIT in most patients. A high IgG/IgE ratio correlated with low skin test reactivity after ≥3 years of VIT., Conclusions: The correlation between a high venom-specific IgG/IgE ratio and low skin test reactivity after VIT may be interesting for future investigations that assess its role as a potential marker for VIT efficacy., (© 2017 S. Karger AG, Basel.)

Parasitization by an endoparasitoid wasp, Cotesia plutellae, alters host development of Plutella xylostella by extending larval period and preventing metamorphosis. Insulin signal plays a crucial role in mediating insect development and controlling blood sugar level in insects. In this study, three insulin-like peptide genes (PxILP1-3) were predicted from the genome of P. xylostella. However, only PxILP1 was confirmed to be expressed in P. xylostella. Starvation suppressed the expression level of PxILP1 and up-regulated plasma trehalose level. RNA interference against PxILP1 mimicked starvation effect and extended the larval period of P. xylostella. Parasitized larvae exhibited significantly lower levels of PxILP1 expression compared to nonparasitized larvae. Injection of wasp-symbiotic polydnavirus C. plutellae bracovirus (CpBV) also suppressed PxILP1 expression and extended the larval period. Injection of a viral segment (CpBV-S30) containing a viral histone H4 (CpBV-H4) also suppressed PxILP1 expression. Co-injection of CpBV-S30 and double-stranded RNA (dsCpBV-H4) specific to CpBV-H4 rescued the suppression of PxILP1 expression. Injection of CpBV-S30 significantly extended larval development. Co-injection of CpBV-S30 with dsCpBV-H4 rescued the delay of larval development. Injection of a bovine insulin to parasitized larvae prevented parasitoid development. These results indicate that parasitism of C. plutellae can down-regulate host insulin signaling with the help of parasitic factor CpBV-H4., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Despite the common occurrence of hymenopteran stings worldwide, primary neurologic manifestations including stroke are rare. We report a case of a healthy male who developed a right middle cerebral artery (MCA) territory ischemic stroke after getting stung by a wasp., Case Report: A 44-year-old man with hypertension presented to the hospital with sudden-onset left hemiparesis, left facial weakness, and dysarthria after being stung by a wasp. Magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) scans of the brain revealed a right MCA territory infarct and a lack of flow in the distal right internal carotid artery and MCA. He was treated with intravenous tissue plasminogen activator. A computed tomography angiography scan of the brain performed 24 hours later revealed multiple regions of vasoconstriction in the territory of the bilateral MCA. Evaluations for causes of stroke, including echocardiography and telemetry, were not revealing. Immunologic testing showed significantly elevated levels of serum wasp immunoglobulin E. Therapy with aspirin and atorvastatin was started. At discharge, the patient had a mild left facial droop but normal strength in his left arm and leg. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians encounter large numbers of hymenopteran sting cases each year. These patients typically present with local reactions, such as itching, pain, and erythema. Systemic manifestations, such as anaphylaxis causing severe hypotension and bronchospasm, are less common but deadly. Neurologic complications, such as ischemic stroke, are extremely rare. This manuscript highlights the pathophysiology and management of stroke after a hymenopteran sting. There are no guidelines for the management of stroke after a hymenopteran sting, and therefore we intend to provide some guidance to physicians for treating stroke after a hymenopteran sting., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bee and wasp stings can cause allergic reactions. Although the local reactions are more frequent, anaphylaxis due to insect stings can be potentially fatal. Rapid recognition of anaphylaxis is therefore critical and reactions should immediately be treated with i.m. adrenaline. Patients having experienced anaphylaxis should be referred to an allergist for diagnostic evaluation and possible venom-immunotherapy (VIT). The clinical history is essential in diagnosis of venom allergy as the test results are not always reliable. Diagnostic testing with venom components might be beneficial in appropriate patients. The analysis of serum tryptase from the acute episode can be crucial. Mastocytosis is associated in about 8 percent of patients with severe anaphylaxis from insect stings and should be considered in the differential diagnosis. VIT is indicated for patients with a history of anaphylaxis and is effective in preventing future anaphylaxis from Hymenoptera stings.

The high frequency of interactions amongst closely related individuals in social insect colonies enhances pathogen transmission. Group-mediated behavior supporting immune defenses tends to decrease selection acting on immune genes. Along with low effective population sizes this might result in relaxed constraint and rapid evolution of immune system genes. Here, we show that antiviral siRNA genes show high rates of molecular evolution with argonaute 2, armitage and maelstrom evolving faster in social bumblebees compared to their socially parasitic cuckoo bumblebees that lack a worker caste. RNAi genes show frequent positive selection at the codon level additionally supported by the occurrence of parallel evolution. Their evolutionary rate is linked to their pathway specific position with genes directly interacting with viruses showing the highest rates of molecular evolution. We suggest that higher pathogen load in social insects indeed drives the molecular evolution of immune genes including antiviral siRNA, if not compensated by behavior., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.