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In New Zealand's ancient Fuscospora spp. or beech forests, two invasive Vespula social wasps Vespula vulgaris (L.) and Vespula germanica (F.) have become significant problems, adversely affecting native birds and invertebrate biodiversity. The nature of chemical communication in these two species is poorly understood, and this work was undertaken to identify the behaviourally active compounds in the venom of the common wasp, Vespula vulgaris (L.). Venom was removed from the stings of both workers and females and analyzed by coupled gas chromatography/electroantennographic detection (GC/EAD) and gas chromatography/mass spectrometry (GC/MS). Two compounds were present in the venom that consistently elicited EAD responses from the antennae of males and workers. Mass spectrometry analysis and syntheses of candidate structures revealed the structures to be N-(3-methylbutyl)acetamide (MBA) and N-(3-methylbutyl)butanamide (MBB). Gyne venom contains significantly larger amounts of MBA and MBB than worker venom. When these two compounds were tested in the field individually or as binary blends in combination with the known food odour (honeydew volatiles), only N-(3-methylbutyl)butanamide or blends containing this compound showed a strong repellent effect on workers to honeydew volatiles at all doses tested. This is the first report of the occurrence of N-(3-methylbutyl)butanamide in nature and the third amide to be identified in the venom of any social wasp. In addition, this work is the first to report the chemical analysis of the venom of V. vulgaris gyne. The repellency effect observed in this study of the venom compound suggests that our definition and understanding of the function of the alarm pheromone need to be reassessed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Due to its huge invasion potential and specialization in honeybee predation, the invasive hornet Vespa velutina nigrithorax represents a high-concern species under both an ecological and economical perspective. In light of the development of specific odorant attractants to be used in sustainable control strategies, we carried out both behavioural assays and chemical analyses to investigate the possibility that, in the invasive population of V. velutina nigrithorax, reproductive females emit volatile pheromones to attract males, as demonstrated in a Chinese non-invasive population. We focused on the secretions produced by sternal and venom glands; because of the volatility and complexity of their composition, both of them could potentially allow an attraction and a species-specific response, decreasing therefore non-target species by-catches. Results of chemical analyses and behavioural assays showed that venom volatiles, although population-specific, are unlikely candidates as male attractants since they do not differ in composition or in quantity between reproductive females and workers and do not attract males. Conversely, sternal gland secretion differs between female castes for the presence of some ketoacids exclusive of gynes already reported as sex pheromones for the non-invasive subspecies V. velutina auraria. Despite such a difference, males are attracted by the sternal gland secretion of both workers and gynes. These results provide a first step to understand the reproductive biology of V. velutina nigrithorax in its invasive range and to develop effective and sustainable management strategies for the species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although the importance of parasitoids as biocontrol agents has long been recognized, systematic studies of the physiological mechanisms are scarce, especially in those parasitoids that are able to successfully invade their hosts by activating host immune responses. This study explored this phenomenon by investigating the effects of ovarian fluid, venom and egg surface characteristics of Tetrastichus brontispae (Hymenoptera: Eulophidae) on host immunity. The results showed that the injection of venom alone induced higher phenoloxidase activity, while a mixture of ovarian plus venom fluids provoked higher granulocyte and plasmatocyte spreading ratios, highlighting the role that egg surface characteristics may play in successful parasitism. After thorough investigation, the presence of a hemomucin homologue was documented on the egg surface (which was named Tetrastichus brontispae adipocyte plasma membrane associated protein-like, TbAPMAP-like), while the absence of polydnaviruses, fibrous layers and virus-like filaments was confirmed. The higher encapsulation index of eggs incubated with TbAPMAP-like polyclonal antibody demonstrated the protection of the protein against encapsulation. These results contribute to our understanding of the mechanisms used by endoparasitoids to evade encapsulation during the early parasitism stage while enriching our knowledge of local active regulatory mechanisms. It is likely that this is the first study to determine the egg protective properties of TbAPMAP-like in host-parasite systems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Communication in ants is based to a great extent on chemical compounds. Recognition of intruders is primarily based on cuticular hydrocarbon (CHC) profile matching but is prone to being cheated. Eucharitid wasps are specific parasitoids of the brood of ants; the immature stages are either well integrated within the colony or are protected within the host cocoons, whereas adult wasps at emergence must leave their host nest to reproduce and need to circumvent the ant recognition system to escape unscathed. The behavioral interactions between eucharitid wasps and workers of their host, the Neotropical ant Ectatomma tuberculatum, are characterized. In experimental bioassays, newly emerged parasitoids were not violently aggressed. They remained still and were grabbed by ants upon contact and transported outside the nest; host workers were even observed struggling to reject them. Parasitoids were removed from the nest within five minutes, and most were unharmed, although two wasps (out of 30) were killed during the interaction with the ants. We analyzed the CHCs of the ant and its two parasitoids, Dilocantha lachaudii and Isomerala coronata, and found that although wasps shared all of their compounds with the ants, each wasp species had typical blends and hydrocarbon abundance was also species specific. Furthermore, the wasps had relatively few CHCs compared to E. tuberculatum (22-44% of the host components), and these were present in low amounts. Wasps, only partially mimicking the host CHC profile, were immediately recognized as alien and actively removed from the nest by the ants. Hexane-washed wasps were also transported to the refuse piles, but only after being thoroughly inspected and after most of the workers had initially ignored them. Being recognized as intruder may be to the parasitoids' advantage, allowing them to quickly leave the natal nest, and therefore enhancing the fitness of these very short lived parasitoids. We suggest that eucharitids take advantage of the hygienic behavior of ants to quickly escape from their host nests., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Endoparasitoid wasps develop at the expense of other insects, leading to their death. Eggs deposited inside the host body induce an immune response, which results in the formation of a melanized cellular capsule around the egg. To evade or counteract this response, endoparasitoids have evolved different strategies, the most often reported being injection into the host of immunosuppressive factors, notably venom proteins, along with the egg. The analysis of venom components has been performed independently in species of different taxa, but the present picture is far from complete. Intriguingly, the question of the level of venom variability inside species has been neglected, although it may partly determine the potential for parasitoid adaptation. Here, we present a short review of our present knowledge of venom components in endoparasitoids, as well as of the only well-known example of intraspecific variability in a venom immune suppressive protein being responsible for variation in parasitoid virulence. We then present data evidencing inter-individual variation of venom protein profiles, using a gel electrophoresis approach, both in laboratory strains and field populations of a figitid and a braconid species. Whether occurrence of such variability may permit a selection of parasitoid venom components driven by the host remains to be tested, notably in the context of the production and use of biological control auxiliaries., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Maternal factors of female wasps that are injected into hosts with their eggs at oviposition play a major role in strategies used by insect parasitoids to overcome host immunity, and to regulate host physiology during early stages of parasitism. Here, we attempted to precisely determine and compare the protein patterns injected by the endoparasitoid Aphidius ervi into two different host systems. Chitosan beads of aphid size designed as artificial and physiologically inert hosts were used as oviposition medium, to be compared with the natural aphid host as young nymphs of Macrosiphum euphorbiae. Proteins that the A. ervi wasp injects into hosts at oviposition were separated by SDS-PAGE, complemented with proteomic techniques. Analyses confirm the identification of A. ervi γ-glutamyl transpeptidase (γ-GT) as a key component in the venom of the endoparasitoid. Using proteomic techniques, the quantity of γ-GT injected by the A. ervi wasp into aphids along with the egg was estimated as approximately 4ng per oviposition strike. We suggest that similar quantities suffice to explain natural parasitization success in A. ervi, which do not rely on polydnavirus to establish into hosts. Moreover, an enolase that showed a high level of sequence identity with teratocyte A. ervi enolase was detected both in chitosan beads extracts, and in extracts of mature eggs excised from the A. ervi ovaries, but not in its venom glands extracts. Detecting enolase shortly after oviposition in the artificial inert hosts at a stage of parasitism when the A. ervi egg is still in the primary chorionated undifferentiated stage suggests the enolase as a chorionic protein of the mature egg. The possible functions of this enolase enzyme for the establishment and early development of A. ervi in aphid hosts are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Long- and short-range sex pheromones appear to play a crucial role in the mate finding and courtship behaviour of most parasitic Hymenoptera. Yet these parasitoids have been rarely investigated and only a few pheromones have been identified. Recent studies have shown that sexual communication of Trissolcus brochymenae (Hymenoptera: Platygastridae), a quasi-gregarious egg parasitoid of the harlequin bug, Murgantia histrionica (Heteroptera: Pentatomidae), starts before contact between males and females when chemical compounds produced by virgin females trigger the courtship behaviour of males. In the present study, the pheromone components involved in the short-range recognition of T. brochymenae females by males were investigated using electrophysiological and behavioural methods. Female body extracts were analyzed through EAG and GC-EAD and the active compounds were identified through GC-MS. The behavioural responses of virgin males to the GC-EAD active compounds were subsequently evaluated in closed arena bioassays. Two active compounds in EAG and behavioural tests, tetradecyl acetate and (Z)-11-hexadecen-1-yl acetate, were identified as sex pheromone components. Both compounds triggered intense male antennation and mount when applied to solvent-washed female cadavers. Dose-response tests showed different curves for the two compounds. This is the first study on the identification of sexual pheromones in Platygastridae., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Metabolic rate, a physiological trait closely related to fitness traits, is expected to evolve in response to two main environmental variables: (1) climate, low metabolic rates being found in dry and hot regions when comparing populations originating from different climates in a common garden experiment and (2) resource limitations, low metabolic rates being selected when resources are limited. The main goal of this study was to investigate if differences in intrinsic resource limitations may have disrupted the expected evolution of metabolic rate in response to climate in a parasitic wasp. We compared CO(2) production of females from 4 populations of a Drosophila parasitoid, Leptopilina boulardi, as an estimate of their metabolic rate. Two populations from a hot and dry area able to synthesise lipids de novo at adult stage were compared with two populations originating from a mild and humid climate where no lipid accumulation during adult life was observed. These last females are thus more limited in lipids than the first ones. We observed that a high metabolic rate has been selected in hot and dry environments, contrarily to the results of a great majority of studies. We suggest that lipogenesis occurring there may have allowed the selection of a higher metabolic rate, as females are less limited in energetic resources than females from the mild environment. A high metabolic rate may have been selected there as it partly compensates for the long distances that females have to cross to find laying opportunities in distant orchards. We suggest that intrinsic resources should be integrated when investigating geographical variations in metabolism as this factor may disrupt evolution in response to climate., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sociality is associated with an increased risk of disease transmission and one of the first defense of the insect colonies is represented by antimicrobial secretions. In many eusocial hymenopteran species venom glands represent one of the most important source of antimicrobial substances. It is known that in highly eusocial species the venom is spread on both the cuticle of insects and the comb, thus becoming a component of the so called "social immunity". So far, it is never been ascertained whether this phenomenon is also present in more primitively eusocial and incipiently eusocial groups. Using incipiently eusocial hover wasps as model, we demonstrate that venom is present on insect cuticles and that it strongly acts against microorganisms. By contrast, the nest, regardless of materials, does not represent a ''medium" where the venom is deposited by wasps in order to act as a social antiseptic weapon. Our findings discussed in an evolutionary perspective indicate that a certain degree of sociality or a sufficient number of individuals in an insect society are thresholds to be reached for the rise of complex and efficient forms of collective and social immunity as mechanisms of resistance to diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Queens of the primitively eusocial wasp Ropalidia marginata appear to maintain reproductive monopoly through pheromone rather than through physical aggression. Upon queen removal, one of the workers (potential queen, PQ) becomes extremely aggressive but drops her aggression immediately upon returning the queen. If the queen is not returned, the PQ gradually drops her aggression and becomes the next queen of the colony. In a previous study, the Dufour's gland was found to be at least one source of the queen pheromone. Queen-worker classification could be done with 100% accuracy in a discriminant analysis, using the compositions of their respective Dufour's glands. In a bioassay, the PQ dropped her aggression in response to the queen's Dufour's gland macerate, suggesting that the queen's Dufour's gland contents mimicked the queen herself. In the present study, we found that the PQ also dropped her aggression in response to the macerate of a foreign queen's Dufour's gland. This suggests that the queen signal is perceived across colonies. This also suggests that the Dufour's gland in R. marginata does not contain information about nestmateship, because queens are attacked when introduced into foreign colonies, and hence PQ is not expected to reduce her aggression in response to a foreign queen's signal. The latter conclusion is especially significant because the Dufour's gland chemicals are adequate to classify individuals correctly not only on the basis of fertility status (queen versus worker) but also according to their colony membership, using discriminant analysis. This leads to the additional conclusion (and precaution) that the ability to statistically discriminate organisms using their chemical profiles does not necessarily imply that the organisms themselves can make such discrimination., (Copyright © 2010 Copyright © 2010 Elsevier Ltd. All rights reserved.)

The egg-larval parasitoid Chelonus inanitus injects bracoviruses (BVs) and venom along with the egg into the host egg; both components are essential for successful parasitoid development. All stages of eggs of its natural host, Spodoptera littoralis, can be successfully parasitized, i.e. from mainly a yolk sphere to a fully developed embryo. Here, we show that the venom contains at least 25 proteins with masses from 14kDa to over 300kDa ranging from acidic to basic. The majority is glycosylated and their persistence in the host is short when old eggs are parasitized and much longer when young eggs are parasitized. Physiological experiments indicated three different functions. (1) Venom synergized the effect of BVs in disrupting host development when injected into third instar larvae. (2) Venom had a transient paralytic effect when injected into sixth instar larvae. (3) In vitro experiments with haemocytes of fourth instar larvae suggested that venom alters cell membrane permeability. We propose that venom promotes entry of BVs into host cells and facilitates placement of the egg in the embryo's haemocoel when old eggs are parasitized. The multifunctionality of the venom might thus be essential in enabling parasitization of all stages of host eggs., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star (DE3) cells was induced by the addition of 0.5mM IPTG. Cultures were grown at 24 and 37 degrees C, and VPr1 more readily partitioned into the soluble fraction at 24 degrees C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

Oogenesis of the parasitoid wasp Eupelmus vuilleti is known to be dependent on host availability. However, examination of ovarian dynamics by microscopy showed that oogenesis and vitellogenesis are initiated before female eclosion and proceed 1-2 days after, independent of host presence. Oogenesis continued beyond the 2nd day only in the presence of hosts, otherwise it was replaced by egg resorption. It is thus possible to distinguish between host-independent and host-dependent periods of oogenesis. In the presence of host, each ovariole (three per ovary) contained generally three oocytes: a fully mature oocyte, a nearly mature one and an immature one. However, host deprived-females resorbed their most mature and their smallest oocytes, but kept one almost mature oocyte per ovariole. Comparison of zero, short and long host deprivation periods showed that females always had the ability to quickly lay eggs to exploit any new host. However, increased deprivation led to a reduction in the number and the viability of eggs. Enzymo-immunological measurements of ecdysteroids were made in whole females, in dissected ovaries and in newly laid eggs. Our results indicated that ecdysteroids play a major role as circulating hormones involved in the regulation of oogenesis.

Pimpla hypochondriaca venom is complex and contains a number of different proteins and polypeptides that exert a variety of effects on insect physiology. In particular, it possesses factors with potent anti-haemocyte and immunosuppressive properties. In the current work, we describe the biochemical isolation of a single venom factor with insect haemocyte anti-aggregation properties. The protein was isolated using gel filtration and ion exchange chromatography, in conjunction with a qualitative in vitro haemocyte anti-aggregation assay to monitor activity and confirm identity. The protein has a molecular weight estimate of 33kDa (determined by SDS PAGE under reducing conditions), and an N-terminal sequence of Asp-Ser-Asp-Ile-Tyr-Leu-Leu. The biochemically isolated protein has been demonstrated to inhibit haemocyte aggregation and to suppress encapsulation responses, using in vitro and in vivo assays, respectively. Furthermore, its gene has been identified as vpr3. The work is presented within the context of the role of P. hypochondriaca venom and the isolated protein in host immune suppression.

Both avian and insect cuckoos must trick their hosts into accepting foreign eggs. In birds this is achieved through egg mimicry. Within the hornets the only known social parasite is the rare Vespa dybowskii. We investigated how the V. dybowskii queen induces tens or hundreds of host workers to accept her eggs and offspring. Since hydrocarbons function as recognition cues in social insects, we investigated these compounds from the surface of eggs and workers of V. dybowskii, both host species (V. simillima and V. crabro) and an additional four non-host species. We found that chemical mimicry of the hosts' colony odour and their eggs normally associated with wasps was not being employed by V. dybowskii. Chemical insignificance is also unlikely as the amounts of hydrocarbons extracted from parasite, host and non-host eggs were similar. Eggs of V. dybowskii may survive in part due to being chemically transparent, as methyl-branched compounds only represent a tiny proportion (<1%) of the parasites hydrocarbon profile but a large proportion (26-41%) in both host species. However, the functions of various hydrocarbon groups need to be investigated in the hornets before this new acceptance mechanism of parasite eggs and adults is understood.

We analysed changes in cuticular hydrocarbon signatures of workers in orphaned colonies of the paper wasp Polistes dominulus. In natural conditions, workers and foundresses possess characteristic cuticular signatures, and foundresses are further distinguishable, both behaviourally and chemically, on the basis of their rank in a reproductive dominance hierarchy. In our study, several workers were found to develop their ovaries and produce cuticular signatures resembling those of dominant foundresses, while remaining workers possessed undeveloped ovaries and had cuticular blends characteristic of subordinate foundresses. Workers that did not develop their ovaries had changed epicuticular signatures, demonstrating that the mixture of hydrocarbons of worker individuals is strongly dependent on social role and environment. Our results suggest that the composition of epicuticular lipids is not determined at the pre-imaginal stage, and that physiological pathways leading to cuticular chemical changes are similar in foundresses and workers of P. dominulus.

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.

In multiple-foundress nests of the wasp Polistes dominulus, dominance hierarchies are established among foundresses, and only the dominant (=alpha) individual lays eggs. The alpha female can be distinguished from subordinate females and workers on the basis of the proportions of some hydrocarbons present on the cuticle, suggesting that chemical signaling of her reproductive status could occur. P. dominulus is also the host species of the obligate social parasite Polistes sulcifer. After aggressively usurping host colonies and behaviorally replacing the host alpha female, parasites are characterized by a change in the proportions of their cuticular hydrocarbons to match that of the host cuticular profile at both species and colony levels. In the current study, we demonstrate that P. sulcifer queens also modify their cuticular hydrocarbon proportions after usurpation to match that of the host alpha female. Parasite females, therefore, acquire the dominant rank in host colonies both reproductively and chemically by mimicking the typical alpha profile of the host. Parasite females were not able to fully inhibit ovary development in host foundresses, and 10 days after usurpation, parasites, alpha and beta foundresses show similar chemical profiles and ovarian development.

In many social insects the relationship between reproductive dominance and physiological correlates is poorly understood. Recent evidence now strongly suggests that cuticular hydrocarbons are important in reproductive differentiation in these societies where they are used as signals of ovarian activity in reproductive females. In this study we investigated the relationship between reproductive dominance, size of the corpora allata (CA, producer of Juvenile Hormone, JH) and the proportions of cuticular hydrocarbons present on the cuticle in overwintering foundresses and both associative (polygynous) and solitary (monogynous) pre-emergence colonies of the social wasp Polistes dominulus. Size of the CA was positively correlated with ovarian development in polygynous colonies. In contrast, solitary foundresses possessed significantly smaller CAs than dominant foundresses from polygynous nests, yet ovarian activity was similar for both female types. CA size variation was associated with variation in cuticular hydrocarbon proportions. Overwintering, solitary, dominant and subordinate (from associative nests) females all possessed distinctive cuticular chemical profiles revealed by multivariate discriminant analyses. Our data indicate that the social environment strongly affects reproductive physiology in this wasp, and we discuss the role of cuticular hydrocarbons in reproductive signaling in P. dominulus and other social insects.