Female volatiles as sex attractants in the invasive population of Vespa velutina nigrithorax.

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.

Wide-scale analysis of protein expression in head and thorax of Aedes albopictus females.

The recently available genome of Aedes albopictus - the most worldwide-spread human arbovirus vector - has revealed a large genome repertory and a great plasticity which are believed to have contributed to the species success as an invasive species and opened the way to genomic, transcriptomic and proteomic studies. We carried out the first wide-scale quantitative proteomic analysis of Ae. albopictus female head and thorax by means of a 'shotgun' approach based on nano liquid chromatography-high resolution mass spectrometry associated to protein Label Free Quantification (LFQ) which allows to assess differences in protein expression between tissues and different physiological stages. We identified 886 and 721 proteins in heads and thoraxes respectively, 5 of which were exclusively expressed in thoraxes and 170 in heads, consistently with the more complex head physiology. Head-protein expression was found to be highly divergent between virgin and mated females and limited before and after blood-feeding and oviposition. The large repertoire of proteins identified represents an instrumental source of data for genome annotation and gene-expression studies, and may contribute to studies aimed at investigating the molecular bases of physiological processes of this successful invasive species.

Candidate biomarkers for mosquito age-grading identified by label-free quantitative analysis of protein expression in Aedes albopictus females.

We applied a "shotgun" approach based on nanoliquid chromatography-high resolution mass spectrometry associated to label free quantification (LFQ) to identify proteins varying with age, independently from the physiological state, in Aedes albopictus, a mosquito species which in the last decades invaded temperate regions in North America and Europe, creating concerns for associated high nuisance and risk of arbovirus transmission. The combined "shotgun" and LFQ approach was shown to be highly suitable to simultaneously compare several biological samples, as needed in a study aimed to analyze different age-groups and physiological states of adult mosquito females. The results obtained represent the first wide-scale analysis of protein expression in Ae. albopictus females: >1000 and 665 proteins were identified from few micrograms of crude protein extracts of mosquito heads and thoraxes, respectively. Six of these proteins were shown to significantly vary from 2- to 16-day-old females, independently from their physiological state (i.e. virgin, mated, host-seeking, blood-fed, and gravid). BIOLOGICAL SIGNIFICANCE: Mosquito-borne diseases, such as malaria, dengue and other arboviroses, are a persistent cause of global mortality and morbidity, affecting hundreds of thousands of people. Billions of people living in tropical areas are at risk of being bitten every day by an infective mosquito female and the spread of tropical species such as Aedes albopictus to temperate areas is creating alarm in the northern hemisphere. Mosquito longevity is a critical factor affecting mosquito-borne pathogen transmission cycles and the mosquito capacity to transmit pathogens. However, large scale analyses of the age structure of mosquito field populations is hampered by the lack of optimal age-grading approaches. Our findings open new perspectives for the development of reliable, simple and cheap protein-based assays to age-grade Ae. albopictus females and, most likely, other mosquito species of higher medical relevance, such as the main dengue vector, Aedes aegypti, and the major Afrotropical malaria vectors. These assays would greatly contribute to epidemiological studies aimed at defining the actual vectorial capacity of a given mosquito species. Moreover, they would be very valuable in assessing the effectiveness of mosquito control interventions based on the relative ratio between young and old individuals before and after the intervention.

Comparative analysis of epicuticular lipid profiles of sympatric and allopatric field populations of Anopheles gambiae s.s. molecular forms and An. arabiensis from Burkina Faso (West Africa).

We analysed by gas chromatography-mass spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) the epicuticular lipid profiles of field females of the major Afro-tropical malaria vector, Anopheles gambiae. The samples were collected in three villages in Burkina Faso (West Africa), where An. gambiae M and S molecular forms and An. arabiensis live sympatrically. The aim was to compare the cuticular hydrocarbon (CHC) composition of individual field specimens of these three taxa, to highlight possible differences among them. All the samples analysed by GC-MS (55 individuals and eight pools) were characterized by the same 48 CHCs and 10 oxygenated compounds. The 19 most abundant CHCs were quantified in 174 specimens by GC-FID: quantitative intra-taxon differences were found between allopatric populations of both An. arabiensis and S-form. Inter-taxa quantitative differences in the relative abundances of some hydrocarbons between pairs of sympatric taxa were also found, which appear to be mainly linked to local situations, with the possible exception of diMeC(35) between An. arabiensis and S-form. Moreover, MeC(29) shows some degree of differentiation between S- and M-form in all three villages. Possible causes of these differences are discussed.

Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera.

The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs.

Soluble proteins of chemical communication in the social wasp Polistes dominulus.

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.

Recognition of social parasites as nest-mates: adoption of colony-specific host cuticular odours by the paper wasp parasite Polistes sulcifer.

Colonies of the polistine wasp Polistes dominulus are parasitized by the permanent worker-less social parasite Polistes sulcifer. After usurpation of the host colony, parasite females are characterized by a change in the relative proportions of their cuticular hydrocarbons to match those of the host species. In this paper we present evidence from field data and laboratory experiments that P. sulcifer females adopt a colony-specific host odour that facilitates their acceptance by host females of the usurped colony. Presentation experiments demonstrate that parasite females are recognized as foreign individuals by workers of other parasitized nests. We show that the modification of parasite cuticular compounds is sufficient for this recognition. This provides evidence that, after invasion, P. sulcifer queens do not require appeasement or propaganda substances for their acceptance by host colonies. Furthermore, multivariate discriminant analysis of the cuticular hydrocarbon proportions of the parasites after usurpation assigns the parasites together with P. dominulus females of their own host colony. To the authors' knowledge, this is the first confirmation that social parasites adopt colony-specific host odours.

(Z)-3-hexenyl (R)-3-hydroxybutanoate: a male specific compound in three North American decorator wasps Eucerceris rubripes, E. conata and E. tricolor.

The males of the decorator wasps Eucerceris have been observed to display abdomen-dragging behavior on plants surrounding their nest. It is thought that this applies a territorial-marking sex pheromone that serves to alert females to the males' territory for courtship and mating. The extracts of three species E. rubripes, E. conata, and E. tricolor have been analyzed by GC-MS. The gas chromatograms revealed the presence, in large quantities, of one common volatile compound in the male head extracts. By analogy with the closely related bee wolves Philanthus, we believe the pheromone is produced in the mandibular glands. Utilizing mass spectrometry, microreactions, FT-IR, and 1H and 13C NMR spectroscopy the structure of the pheromone was proposed to be (Z)-3-hexenyl 3-hydroxybutanoate, which was confirmed by synthesis. The absolute configuration of the chiral center was determined to be R for the three species by preparing the Mosher esters of the insect samples and comparing their GC retention times with a synthetic sample of known absolute configuration. In addition 2- and 3-hexenoic acid and some aromatic compounds were also found in varying quantities in both males and females along with hydrocarbons and fatty acids, although no species-specific profiles emerged.

Nestmate recognition in Parischnogaster striatula (Hymenoptera Stenogastrinae), visual and olfactory recognition cues.

The recognition of nestmates from alien individuals is a well known phenomenon in social insects. In the stenogastrine wasp Parischnogaster striatula, we investigated the ability of females to recognize nestmates and the cues on which such recognition is based. Recognition of nestmates was observed in naturally occurring interactions between wasps approaching a nest and the resident females on that nest. This recognition was confirmed in experiments in which nestmates or alien conspecifics were presented to resident females. In naturally occurring interactions, nestmates generally approach their nest with a direct flight, while aliens usually hover in front of the nest before landing. In experiments in which the presented wasps were placed close to the nest in a direct manner, antennation of the presented wasp generally occurred, indicating that chemical cues are involved. Experiments in which dead alien individuals, previously washed in hexane, and then reapplied with extracts were recognized by colonies giving further evidence that chemical cues mediate nestmate recognition. Epicuticular lipids, known to be nestmate recognition cues in social insects, were chemically analysed by GC-MS for 44 P. striatula females from two different populations (13 different colonies). Discriminant analysis was performed on the data for the lipid mixture composition. The discriminant model showed that, in the samples from these two populations, 68.2% and 81.9% of the specimens could be correctly assigned to their colony.

Social hackers: integration in the host chemical recognition system by a paper wasp social parasite.

Obligate social parasites in the social insects have lost the worker caste and the ability to establish nests. As a result, parasites must usurp a host nest, overcome the host recognition system, and depend on the host workers to rear their offspring. We analysed cuticular hydrocarbon profiles of live parasite females of the paper wasp social parasite Polistes sulcifer before and after usurpation of host nests, using the non-destructive technique of solid-phase micro-extraction. Our results reveal that hydrocarbon profiles of parasites change after usurpation of host nests to match the cuticular profile of the host species. Chemical evidence further shows that the parasite queen changes the odour of the nest by the addition of a parasite-specific hydrocarbon. We discuss the possible role of this in the recognition and acceptance of the parasite and its offspring in the host colony.

Chemistry, ontogeny, and role of pygidial gland secretions of the vinegaroon Mastigoproctus giganteus (Arachnida: Uropygi).

Pygidial gland secretions of the vinegaroon, Mastigoproctus giganteus, consist of 26% water and 74% short-chained fatty acids, with acetic acid and octanoic acids the dominant acids. The organic composition of the secretion consists of 83% acetic acid, 15% octanoic acid, 1% (Z)-5-octenoic acid, 0.0002% of the corresponding (E)-isomer, 0.11% heptanoic acid, 0.02% hexanoic acid, 0.007% decanoic acid, and in some individuals a trace of 2-nonanone. Formic acid is absent from the secretion. Glandular composition, especially in the concentrations of the minor components, varies dramatically among individuals and shows no age pattern differences among the four free-living subadult instars, or sex differences between adult males and females. Each individual exhibits its own specific blend of components which is identical in the two separate and different sized glands. The secretions act solely as allomones against potential predators, and do not act as pheromones, or elicit a communicative or behavioral response. Behavioral tests using live animals or a synthetic blend of vinegaroon secretion revealed that the secretion is maximally effective on sensitive respiratory, visual, or sensory systems of potential predators and is ineffective, or nearly so, when contact is restricted to inside the predator's mouth. Thus, the effectiveness of the secretion apparently results from its delivery as a spray of fine droplets, rather than as an exudate or vapor.

Chemical analysis of the swarming trail pheromone of the social wasp Polybia sericea (Hymenoptera: Vespidae).

The behaviour of those polistine wasps which found their nests by swarming, suggests that these species use trail pheromones for leading the swarm to the location chosen for the new nest. Apart from a recent report regarding the ropalidine Polybioides tabidus, where the pheromone is thought to originate from the Dufour gland, nothing is known about the chemistry of such pheromones. Polybia sericea is the only species for which the source of the trail pheromone, the Richards' gland, is known with certainty. The chemistry of the Richards' gland secretion of this species has been investigated in the present work and shown to be a complex mixture where the major compounds are alkyl and aromatic aldehydes, fatty acids, 3-phenylpropanoic acid, ketones, a macrolactone, a pyranone compound and nerolidol.

Ant repellent effect of the sternal gland secretion ofPolistes dominulus (Christ) andP. sulcifer (Zimmermann). (Hymenoptera: Vespidae).

The long-chain carboxylic acids identified in the sternal gland secretion ofPolistes dominulus andP. sulcifer females were tested individually on three species of ants,Crematogaster scutellaris, Formica cunicularia, andLasius sp., in order to verify if they have a repellent effect. The unsaturated acids (palmitoleic, linoleic, and oleic) act as repellents of all three ant species, while the saturated acids (lauric, myristic, palmitic, and stearic) have no effect. The mixture reproducing the secretion of the sternal glands ofP. dominulus maintained its repellency for at least four days.

Chemical analysis of sternal gland secretion of paper waspPolistes dominulus (Christ) and its social parasitePolistes sulcifer (Zimmermann) (Hymenoptera: Vespidae).

The secretion of the tegumental glands of the fifth and sixth gastral sternites ofP. dominulus and of its social parasiteP. sulcifer was analyzed by gas chromatography-mass spectrometry. Seven long-chain carboxylic acids have been identified: hexadecanoic, octadecadienoic and octadecenoic acids are the major components of the gland secretion ofP. dominulus and octadecenoic acid the main compounds ofP. sulcifer sternal gland secretion. The same carboxylic acids have been found on the nest pedicel.

Abdomen stroking behaviour and its possible functions in Polistes dominulus (christ) (hymenoptera, vespidae).

The behaviour of stroking the abdomen over the nest surface has previously been described in females of only a few Polistes wasp species, and was considered to be associated with pheromonal release. This study examines abdomen stroking in solitary and multiple female laboratory colonies of Polistes dominulus during the pre-emergence period. In multiple female colonies stroking behaviour is related to hierarchy: alpha females stroking more than subordinates. However, after removal of the dominant female the subordinate increases her stroking rate. Solitary females also stroke but, unlike alpha females, they do so less during the egg-stage than in subsequent stages of the colonial cycle. It is hypothesized that abdomen stroking cues the immature brood as to which adult female is the dominant individual; this could lead to competition between females on the same nest. This function is also discussed in the context of intra- and inter-specific Polistes parasites, where the behaviour is particularly evident.