Venom Collection by Electrical Stimulation in the Invasive Species Polistes dominula Reared Using a Vespiculture Regime.

Specific Venom Immunotherapy (VIT) is practiced with venom extracted from insects, and is the specific therapy used for patients highly allergic to social insect (Hymenoptera) stings. Due to the dramatic shortage of vespid species in the local environment, we coupled vespiculture techniques of Polistes paper wasps with a venom collection procedure based on the electrical stimulation of individuals from entire colonies. The procedure involves little to no disturbance of the individual insects, and at the same time, successfully allows for the extraction of venom containing all allergens necessary for VIT.

Antifungals, arthropods and antifungal resistance prevention: lessons from ecological interactions.

Arthropods can produce a wide range of antifungal compounds, including specialist proteins, cuticular products, venoms and haemolymphs. In spite of this, many arthropod taxa, particularly eusocial insects, make use of additional antifungal compounds derived from their mutualistic association with microbes. Because multiple taxa have evolved such mutualisms, it must be assumed that, under certain ecological circumstances, natural selection has favoured them over those relying upon endogenous antifungal compound production. Further, such associations have been shown to persist versus specific pathogenic fungal antagonists for more than 50 million years, suggesting that compounds employed have retained efficacy in spite of the pathogens' capacity to develop resistance. We provide a brief overview of antifungal compounds in the arthropods' armoury, proposing a conceptual model to suggest why their use remains so successful. Fundamental concepts embedded within such a model may suggest strategies by which to reduce the rise of antifungal resistance within the clinical milieu.

Home economics in an oak gall: behavioural and chemical immune strategies against a fungal pathogen in Temnothorax ant nests.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

Social wasps are a Saccharomyces mating nest.

The reproductive ecology of Saccharomyces cerevisiae is still largely unknown. Recent evidence of interspecific hybridization, high levels of strain heterozygosity, and prion transmission suggest that outbreeding occurs frequently in yeasts. Nevertheless, the place where yeasts mate and recombine in the wild has not been identified. We found that the intestine of social wasps hosts highly outbred S. cerevisiae strains as well as a rare S. cerevisiae×S. paradoxus hybrid. We show that the intestine of Polistes dominula social wasps favors the mating of S. cerevisiae strains among themselves and with S. paradoxus cells by providing a succession of environmental conditions prompting cell sporulation and spores germination. In addition, we prove that heterospecific mating is the only option for European S. paradoxus strains to survive in the gut. Taken together, these findings unveil the best hidden secret of yeast ecology, introducing the insect gut as an environmental alcove in which crosses occur, maintaining and generating the diversity of the ascomycetes.

Trans-generational immunization in the acrobat ant Crematogaster scutellaris.

Trans-generational immunization is defined as the transmission of an enhanced resistance to a pathogen from parents to offspring. By using the host-parasite system of the ant Crematogaster scutellaris and the entomopathogenic fungus Metarhizium anisopliae, we describe this phenomenon for the first time in ants. We exposed four groups of hibernating queens to different treatments (i) a non-lethal dose of live conidiospores in Triton, (ii) a dose of heat-killed conidiospores in Triton, (iii) a control Triton solution, and (iv) a naive control. We exposed their first workers to a high dose of conidiospores and measured mortality rates. Workers produced by queens exposed to live conidiospores survived longer than those belonging to the other groups, while exposure to Triton and dead spores had no effect. Starved workers showed a significantly higher mortality. The treatments did not influence queen mortality, nor the number of offspring they produced at the emergence of the first worker, showing no evidence of immunization costs-at least for these parameters in the first year of colony development. We propose that trans-generational immunization represents an important component of social immunity that could affect colony success, particularly during the critical phase of claustral foundation.

Chemical Communication and Reproduction Partitioning in Social Wasps.

Social wasps encompass species displaying diverse social organization regarding colony cycle, nest foundation, caste differences (from none to significant dimorphism) and number of reproductive queens. Current phylogenetic data suggests that sociality occured independently in the subfamily Stenogastrinae and in the Polistinae+Vespinae clade. In most species, including those with the simplest social organization, colony reproduction is monopolised by a single or few females. Since their nest mates can also develop ovaries and lay eggs, dominant females must somehow inhibit them from reproducing. Physical interactions in the form of open aggression or, usually, ritualised dominance by the fertile females contribute to fertility inhibition in several species, but it is unlikely to function in large colonies. In the latter case, reproduction within the colony is likely to be regulated through pheromones. Relatively little is known about these semiochemicals. Studies on all the three social wasp subfamilies, revealed that cuticular hydrocarbon components differ in abundance between egg-laying and not egg-laying females and that their composition depends on fertility status. In several species, females have been reported to manifestly react towards females with activated ovaries, but there is little evidence to support the hypothesis that fertile individuals are either recognized through their CHC composition, or that over-represented CHC constituents can inhibit fertility. Moreover, very little information exists on the possibility that exocrine glands release fertility signals or chemicals inhibiting fertility.

Social wasp intestines host the local phenotypic variability of Saccharomyces cerevisiae strains.

Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess whether the strains found in wasp intestines are characterized by peculiar traits. We analysed strains isolated from the intestines of social wasps and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to representing a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains; their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved, to safeguard not only the natural variance of this microorganism but also the interests of wine-makers, who could take advantage from the exploitation of their phenotypic variability. Copyright © 2016 John Wiley & Sons, Ltd.

Facial patterns in a tropical social wasp correlate with colony membership.

Social insects excel in discriminating nestmates from intruders, typically relying on colony odours. Remarkably, some wasp species achieve such discrimination using visual information. However, while it is universally accepted that odours mediate a group level recognition, the ability to recognise colony members visually has been considered possible only via individual recognition by which wasps discriminate 'friends' and 'foes'. Using geometric morphometric analysis, which is a technique based on a rigorous statistical theory of shape allowing quantitative multivariate analyses on structure shapes, we first quantified facial marking variation of Liostenogaster flavolineata wasps. We then compared this facial variation with that of chemical profiles (generated by cuticular hydrocarbons) within and between colonies. Principal component analysis and discriminant analysis applied to sets of variables containing pure shape information showed that despite appreciable intra-colony variation, the faces of females belonging to the same colony resemble one another more than those of outsiders. This colony-specific variation in facial patterns was on a par with that observed for odours. While the occurrence of face discrimination at the colony level remains to be tested by behavioural experiments, overall our results suggest that, in this species, wasp faces display adequate information that might be potentially perceived and used by wasps for colony level recognition.

Chemical disguise of myrmecophilous cockroaches and its implications for understanding nestmate recognition mechanisms in leaf-cutting ants.

BACKGROUND: Cockroaches of the genus Attaphila regularly occur in leaf-cutting ant colonies. The ants farm a fungus that the cockroaches also appear to feed on. Cockroaches disperse between colonies horizontally (via foraging trails) and vertically (attached to queens on their mating flights). We analysed the chemical strategies used by the cockroaches to integrate into colonies of Atta colombica and Acromyrmex octospinosus. Analysing cockroaches from nests of two host species further allowed us to test the hypothesis that nestmate recognition is based on an asymmetric mechanism. Specifically, we test the U-present nestmate recognition model, which assumes that detection of undesirable cues (non-nestmate specific substances) leads to strong rejection of the cue-bearers, while absence of desirable cues (nestmate-specific substances) does not necessarily trigger aggression. RESULTS: We found that nests of Atta and Acromyrmex contained cockroaches of two different and not yet described Attaphila species. The cockroaches share the cuticular chemical substances of their specific host species and copy their host nest's colony-specific cuticular profile. Indeed, the cockroaches are accepted by nestmate but attacked by non-nestmate ant workers. Cockroaches from Acromyrmex colonies bear a lower concentration of cuticular substances and are less likely to be attacked by non-nestmate ants than cockroaches from Atta colonies. CONCLUSIONS: Nest-specific recognition of Attaphila cockroaches by host workers in combination with nest-specific cuticular chemical profiles suggest that the cockroaches mimic their host's recognition labels, either by synthesizing nest-specific substances or by substance transfer from ants. Our finding that the cockroach species with lower concentration of cuticular substances receives less aggression by both host species fully supports the U-present nestmate recognition model. Leaf-cutting ant nestmate recognition is thus asymmetric, responding more strongly to differences than to similarities.

Role of social wasps in Saccharomyces cerevisiae ecology and evolution.

Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain's evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Mutualistic Relationships between Microorganisms and Eusocial Wasps (Hymenoptera, Vespidae).

Eusocial wasps are represented in the Vespidae by the subfamilies Stenogastrinae, Vespinae and Polistinae. These wasps present colonies that are sometimes composed of thousands of individuals which live in nests built with paper materials. The high density of the adult and larval population, as well as the stable micro environment of the nests, make very favourable conditions for the flourishing of various types of microorganisms. These microorganisms, which may be pathogens, are beneficial and certainly contribute to model the sociality of these insects. The mutualistic relationships that we observe in some species, especially in Actinomycete bacteria and yeasts, could have important fallouts for the development of new medicines and for the use of these insects in agricultural environments.

Using wasps as a tool to restore a functioning vine grape mycobiota and preserve the mycobial "terroir".

In the last one-hundred years, the exponential expansion of wine making has artificialized the agricultural landscape as well as its microbial diversity, spreading human selected Saccharomyces cerevisiae strains. Evidence showed that social wasps can harbor a significant fraction of the yeast phenotypic diversity of a given area of wine production, allowing different strains to overwinter and mate in their gut. The integrity of the wasp-yeast ecological interaction is of paramount importance to maintain the resilience of microbial populations associated to wine aromatic profiles. In a field experiment, we verified whether Polistes dominula wasps, reared in laboratory and fed with a traceable S. cerevisiae strain, could be a useful tool to drive the controlled yeast dispersion directly on grapes. The demonstration of the biotechnological potential of social insects in organic wine farming lays the foundations for multiple applications including maintenance of microbial biodiversity and rewilding vineyards through the introduction of wasp associated microbiomes.

Evaluation and validation of a bee venom sting challenge performed by a micro-syringe.

BACKGROUND: The honeybee sting challenge is considered a reliable procedure to evaluate the efficacy of specific immunotherapy, but it is difficult and unpractical to perform in clinical practice, because live insects are required. OBJECTIVE: To assess the feasibility and reliability of a challenge test using a micro-syringe, and compared the procedure with sting challenge. METHODS: Patients on bee venom immunotherapy and without systemic reactions at field sting were enrolled. They underwent a sting challenge with live bee, and large local reactions were assessed up to 48 hours. Those patients displaying systemic reactions at the sting challenge were excluded from the syringe challenge for ethical reasons. The syringe challenge was done by injecting 0.5 µL fresh unfiltered bee venom at 2 mm depth (the length of the sting left by a bee). The same follow-up as at the first challenge was performed. Bee-specific immunoglobulin E (IgE) and tryptase were measured after each challenge. RESULTS: Nineteen patients underwent the sting challenge with live bees. Four had immediate systemic reactions (urticaria or asthma) and were excluded from the second challenge. The remaining 15 patients with large local reaction underwent the syringe challenge. No significant difference was seen in the maximum area of the large local reactions between the challenge with live bees and the syringe challenge. Also, no change was seen in tryptase and specific antibodies. CONCLUSION: This preliminary study suggests that the micro-syringe challenge with honeybee venom is feasible and produces results indistinguishable from those of the traditional sting challenge.

Effect of climate on strategies of nest and body temperature regulation in paper wasps, Polistes biglumis and Polistes gallicus.

Polistes paper wasps are a widespread taxon inhabiting various climates. They build nests in the open without a protective outer layer, which makes them vulnerable to changing temperatures. To better understand the options they have to react to environmental variation and climate change, we here compare the thermoregulatory behavior of Polistes biglumis from cool Alpine climate with Polistes gallicus from warm Mediterranean climate. Behavioral plasticity helps both of them to withstand environmental variation. P. biglumis builds the nests oriented toward east-south-east to gain solar heat of the morning sun. This increases the brood temperature considerably above the ambience, which speeds up brood development. P. gallicus, by contrast, mostly avoids nesting sites with direct insolation, which protects their brood from heat stress on hot days. To keep the brood temperature below 40-42 °C on warm days, the adults of the two species show differential use of their common cooling behaviors. While P. biglumis prefers fanning of cool ambient air onto the nest heated by the sun and additionally cools with water drops, P. gallicus prefers cooling with water drops because fanning of warm ambient air onto a warm nest would not cool it, and restricts fanning to nests heated by the sun.

Differential expression of odorant-binding proteins in the mandibular glands of the honey bee according to caste and age.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers' glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals.

Cuticular hydrocarbons rather than peptides are responsible for nestmate recognition in Polistes dominulus.

A colony of social insects is like a fortress where access is allowed only to colony members. The epicuticular mixture of hydrocarbons has been widely reported to be involved in nestmate recognition in insects. However, recent studies have shown that polar compounds (mainly peptides) are also present, mixed with hydrocarbons, on the cuticle of various insects, including the paper wasps of the genus Polistes. As these polar compounds are variable among Polistes species and are perceived by the wasps, this cuticular fraction could also be involved in nestmate recognition. Through MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time of Flight) mass spectrometry analysis, we assessed, for the first time, the intercolonial variability of the cuticular polar fraction of Polistes dominulus in order to evaluate its reliability as source of nestmate recognition cues. We then tested through behavioral assays the importance of the 2 isolated fractions (apolar and polar) in nestmate recognition by presenting them separately to colonies of P. dominulus. Our results showed that the cuticular polar compounds are not colony specific and they are not used by paper wasps to discriminate nestmates from non-colony members. On the contrary, we confirmed that the isolated cuticular hydrocarbons are the chemical mediators prompting nestmate recognition in paper wasps.

The chemical basis of host nest detection and chemical integration in a cuckoo paper wasp.

Insect social life is governed by chemicals. A great number of studies have demonstrated that the blend of hydrocarbons present on the cuticle (CHCs) plays a pivotal role in intra- and inter-specific communication. It is not surprising, therefore, that social parasites, specialized in exploiting the costly parental care provided by host workers, exploit the host chemical communication system too. Throughout their life cycle, social parasites intercept and break this CHC-based code. Recently, however, several polar compounds (mainly peptides) have been found in addition to CHCs both on the cuticle and on the comb surface of social insects, and their semiochemical role has been demonstrated in some circumstances. In the present study, we used the paper wasp social parasite-host system Polistes sulcifer (Zimmerman)-Polistes dominulus (Christ) to evaluate the relative importance of the CHCs and polar compounds in two different steps of the host exploitation process: host nest detection by the pre-usurping parasite and parasite chemical integration into the host colony. After separating the polar and apolar fractions of the host nest as well as those of pre- and post-usurpation parasites, we carried out laboratory assays based on the binary choice model. Our results show that nest polar compounds neither are used by the parasite to detect the host's nest nor play a role in parasite chemical integration into the host colony. In contrast, we demonstrate that CHCs are fundamental in both steps, thus confirming their primary role in social insect life and consequently in social parasite-host interactions.

Mapping the expression of soluble olfactory proteins in the honeybee.

Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs.

Social dominance molds cuticular and egg chemical blends in a paper wasp.

Hamilton's theory [1] for the evolution of social behaviour predicts that helpers may renounce direct reproduction to help their more fertile kin. Intra-colony recognition among queens and helpers (subordinate queens or workers) is consequently a central issue in insect sociobiology. In social insects, cuticular hydrocarbons (CHCs) are involved in recognition, and egg-laying and non-egg-laying individuals often differ in CHC composition. These differences are assumed to be directly determined by fertility status [2,3]. In several ants and in Polistes wasps, when egg-layers disappear they are substituted by helpers, which develop their ovaries and become chemically similar to their former queens [2,3]. Sometimes helpers lay eggs in the presence of queens, which recognize and destroy the subordinates' eggs [4]. In ponerine ants, eggs often have the same chemical signature as the maternal cuticle [2]. If chemical signatures depend on fertility, egg-laying subordinates should match the queen's signature even when she is present, making egg recognition and differential oophagy impossible. In the study reported here, we experimentally separated fertility from dominance and analyzed the dynamics of hydrocarbon profiles of the cuticle of Polistes dominulus foundresses and the shell surface of their eggs. We have demonstrated that, contrary to the widely accepted view, dominance, rather than fertility, determines chemical signatures in Polistes wasps. This explains why queens can recognize their own eggs and police reproduction by subordinates if they become fertile and lay eggs.