Unravelling the microplastic menace: Different polymers additively increase bee vulnerability.

Microplastics (MPs) are growing and ubiquitous environmental pollutants and represent one of the greatest contemporary challenges caused by human activities. Current research has predominantly examined the singular toxicological effects of individual polymers, neglecting the prevailing reality of organisms confronted with complex contaminant mixtures and potential synergistic effects. To fill this research gap, we investigated the lethal and sublethal effects of two common MPs, polystyrene (PS - 4.8-5.8 µm) and poly(methyl methacrylate) (PMMA - 1-40 µm), and their combination (MIX), on the pollinating insect Apis mellifera. For each treatment, we evaluated the oral toxicity of two ecologically relevant and one higher concentration (0.5, 5 and 50 mg/L) and analysed their effects on the immune system and worker survival. As immune activation can alter the cuticular hydrocarbon profile of honey bees, we used gas chromatography-mass spectrometry (GC-MS) to investigate whether MPs lead to changes in the chemical profile of foragers and behavioural assay to test whether such changes affect behavioural patterns of social recognition, undermining overall colony integrity. The results indicate an additive negative effect of PS and PMMA on bee survival and immune response, even at ecologically relevant concentrations. Furthermore, alterations in cuticle profiles were observed with both MPs at the highest and intermediate concentrations, with PMMA being mainly responsible. Both MPs exposure resulted in a reduction in the abundance of several cuticular compounds. Hive entry guards did not show increased inspection or aggressive behaviour towards exposed foragers, allowing them to enter the colony without being treated differently from uncontaminated foragers. These findings raise concerns not only for the health of individual bees, but also for the entire colony, which could be at risk if contaminated nestmates enter the colony undetected, allowing MPs to spread throughout the hive.

Putting hornets on the genomic map.

Hornets are the largest of the social wasps, and are important regulators of insect populations in their native ranges. Hornets are also very successful as invasive species, with often devastating economic, ecological and societal effects. Understanding why these wasps are such successful invaders is critical to managing future introductions and minimising impact on native biodiversity. Critical to the management toolkit is a comprehensive genomic resource for these insects. Here we provide the annotated genomes for two hornets, Vespa crabro and Vespa velutina. We compare their genomes with those of other social Hymenoptera, including the northern giant hornet Vespa mandarinia. The three hornet genomes show evidence of selection pressure on genes associated with reproduction, which might facilitate the transition into invasive ranges. Vespa crabro has experienced positive selection on the highest number of genes, including those putatively associated with molecular binding and olfactory systems. Caste-specific brain transcriptomic analysis also revealed 133 differentially expressed genes, some of which are associated with olfactory functions. This report provides a spring-board for advancing our understanding of the evolution and ecology of hornets, and opens up opportunities for using molecular methods in the future management of both native and invasive populations of these over-looked insects.

Bats mimic hymenopteran insect sounds to deter predators.

Mimicry is one of the most fascinating phenomena in nature1. Mimicry traits often reflect complex, finely tuned, and sometimes extravagant relationships among species and have evolved to deceive predators or prey. Indeed, mimicry has most often evolved to discourage predation: the 'mimic' exhibits phenotypic convergence towards a non-related 'model' organism which is inedible or harmful, so that a given predator, or 'receiver', will refrain from attacking or ingesting the mimic. Traditionally, mimicry is mainly evident and has been mainly studied in the visual domain. Here, we report experiments that document the first case of interspecific acoustic mimicry in a mammal and demonstrate that the distress calls the greater mouse-eared bat (Myotis myotis) broadcasts when handled imitate sounds of stinging bees or wasps to discourage the bat's avian predators.

Biopesticides and insect pollinators: Detrimental effects, outdated guidelines, and future directions.

As synthetic pesticides play a major role in pollinator decline worldwide, biopesticides have been gaining increased attention to develop more sustainable methods for pest management in agriculture. These biocontrol agents are usually considered as safe for non-target species, such as pollinators. Unfortunately, when it comes to non-target insects, only the acute or chronic effects on survival following exposure to biopesticides are tested. Although international boards have highlighted the need to include also behavioral and morphophysiological traits when assessing risks of plant protection products on pollinators, no substantial concerns have been raised about the risks associated with sublethal exposure to these substances. Here, we provide a comprehensive review of the studies investigating the potential adverse effects of biopesticides on different taxa of pollinators (bees, butterflies, moths, beetles, flies, and wasps). We highlight the fragmentary knowledge on this topic and the lack of a systematic investigation of these negative effects of biopesticides on insect pollinators. We show that all the major classes of biopesticides, besides their direct toxicity, can also cause a plethora of more subtle detrimental effects in both solitary and social species of pollinators. Although research in this field is growing, the current risk assesment approach does not suffice to properly assess all the potential side-effects that these agents of control may have on pollinating insects. Given the urgent need for a sustainable agriculture and wildlife protection, it appears compelling that these so far neglected detrimental effects should be thoroughly assessed before allegedly safe biopesticides can be used in the field and, in this view, we provide a perspective for future directions.

Hornets and Honey Bees: A Coevolutionary Arms Race between Ancient Adaptations and New Invasive Threats.

Hornets and honey bees have a long history of coevolution resulting in a plethora of captivating adaptations and counteradaptations between predator and prey. From simple physiological mechanisms to complex behavioral strategies, some Vespa hornets have specialized in hunting honey bees, while the latter have put in place effective defenses to counteract their attack. Both hornets and honey bees have evolved the ability to detect the odors and the pheromones emitted by the other to locate the prey or to spot foraging predators. Hornets often rely on their bigger size, heavily armored body and destructive attacks, while honey bees differentiated collective defense responses finely coordinated to deter or kill the hornet menace. However, when new species of hornets and honey bees come into contact, the absence of coevolution can have a heavy impact on the defenseless bees. The evolutionary arms race between hornets and honey bees provides not only compelling examples of adaptations and counteradaptations between predator and prey, but could also represent a starting point for the development of effective and sustainable strategies to protect honey bees and beekeeping activities and to control invasive alien species of hornets.

Cuticular Hydrocarbon Profile of Parasitic Beetles, Aethina tumida (Coleoptera: Nitidulidae).

Cuticular hydrocarbons (CHCs) cover insects' bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs), Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.

A Stresipteran parasite extends the lifespan of workers in a social wasp.

In social wasps, female lifespan depends on caste and colony tasks: workers usually live a few weeks while queens as long as 1 year. Polistes dominula paper wasps infected by the strepsipteran parasite Xenos vesparum avoid all colony tasks, cluster on vegetation where parasite dispersal and mating occur, hibernate and infect the next generation of wasp larvae. Here, we compared the survival rate of infected and uninfected wasp workers. Workers' survival was significantly affected by parasite sex: two-third of workers parasitized by a X. vesparum female survived and overwintered like future queens did, while all workers infected by a X. vesparum male died during the summer, like uninfected workers that we used as controls. We measured a set of host and parasite traits possibly associated with the observed lifespan extension. Infected overwintering workers had larger fat bodies than infected workers that died in the summer, but they had similar body size and ovary development. Furthermore, we recorded a positive correlation between parasite and host body sizes. We hypothesize that the manipulation of worker's longevity operated by X. vesparum enhances parasite's fitness: if workers infected by a female overwinter, they can spread infective parasite larvae in the spring like parasitized gynes do, thus contributing to parasite transmission.

Altered feeding behavior and immune competence in paper wasps: A case of parasite manipulation?

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.

Exposure to a biopesticide interferes with sucrose responsiveness and learning in honey bees.

The entomopathogenic fungus Beauveria bassiana is a widely used biopesticide that is considered as an effective alternative to classical agrochemicals. B. bassiana is thought to be safe for pollinators although little is known about its side-effects on pollinators' behaviour and cognition. Here, we focused on honey bees and used the proboscis extension response (PER) protocol to assess whether B. bassiana affects individual sucrose responsiveness, non-associative and associative olfactory learning and memory. Fungus-treated bees displayed an enhanced sucrose responsiveness, which could not be explained by metabolic alterations. Strikingly, exposed bees were twice as inconsistent as controls in response to sucrose, showing PER to lower but not to higher sucrose concentrations. Exposed bees habituated less to sucrose and had a better acquisition performance in the conditioning phase than controls. Further, neither mid- nor long-term memory were affected by the fungus. As sucrose responsiveness is the main determinant of division of foraging labour, these changes might unsettle the numerical ratio between the sub-castes of foragers leading to suboptimal foraging. Although the use of biocontrol strategies should be preferred over chemical pesticides, careful assessment of their side-effects is crucial before claiming that they are safe for pollinators.

Gut microbial composition in different castes and developmental stages of the invasive hornet Vespa velutina nigrithorax.

Social insects are successful animal invaders. Their survival and success, and in some cases also their impact on invaded ecosystem functioning, is often mediated by symbiosis with microorganisms. Here, we report a comprehensive comparative characterization of the gut microbial communities of different castes and developmental stages of the invasive hornet Vespa velutina nigrithorax. The species recently colonized Europe, becoming a high ecological and economic concern, as it threatens pollinator survival and competes with native hornet species. We used targeted meta-genomics to describe the yeasts and bacteria gut communities of individuals of different reproductive phenotypes (workers and future queens), life stages (larvae, newly emerged individuals and adults) and colony non-living samples (nest paper and larval faeces). Bacilli, Gammaproteobacteria, Actinobacteria, Alphaproteobacteria were the most abundant classes of bacteria, and Saccharomycetes, Dothideomycetes, Tremellomycetes and Eurotiomycetes were the most represented yeast classes. We found that the microbial compositions significantly differ across developmental stages and castes, with yeast and bacterial communities switching in frequency and abundance during ontogeny and according to reproductive phenotype. Moreover, the gut microbial communities poorly mirror those found in the nest, suggesting that hornets possess a specific microbial signature. Our results provide the first metagenomic resource of the microbiome of V. velutina in Europe and suggest the importance of considering life stages, reproductive phenotypes and nest influence in order to obtain a comprehensive picture of social insect microbial communities.

Increased immunocompetence and network centrality of allogroomer workers suggest a link between individual and social immunity in honeybees.

The significant risk of disease transmission has selected for effective immune-defense strategies in insect societies. Division of labour, with individuals specialized in immunity-related tasks, strongly contributes to prevent the spread of diseases. A trade-off, however, may exist between phenotypic specialization to increase task efficiency and maintenance of plasticity to cope with variable colony demands. We investigated the extent of phenotypic specialization associated with a specific task by using allogrooming in the honeybee, Apis mellifera, where worker behaviour might lower ectoparasites load. We adopted an integrated approach to characterize the behavioural and physiological phenotype of allogroomers, by analyzing their behavior (both at individual and social network level), their immunocompetence (bacterial clearance tests) and their chemosensory specialization (proteomics of olfactory organs). We found that allogroomers have higher immune capacity compared to control bees, while they do not differ in chemosensory proteomic profiles. Behaviourally, they do not show differences in the tasks performed (other than allogrooming), while they clearly differ in connectivity within the colonial social network, having a higher centrality than control bees. This demonstrates the presence of an immune-specific physiological and social behavioural specialization in individuals involved in a social immunity related task, thus linking individual to social immunity, and it shows how phenotypes may be specialized in the task performed while maintaining an overall plasticity.

Rethinking recognition: social context in adult life rather than early experience shapes recognition in a social wasp.

Social recognition represents the foundation of social living. To what extent social recognition is hard-wired by early-life experience or flexible and influenced by social context of later life stages is a crucial question in animal behaviour studies. Social insects have represented classic models to investigate the subject, and the acknowledged idea is that relevant information to create the referent template for nest-mate recognition (NMR) is usually acquired during an early sensitive period in adult life. Experimental evidence, however, highlighted that other processes may also be at work in creating the template and that such a template may be updated during adult life according to social requirements. However, currently, we lack an ad hoc experiment testing the alternative hypotheses at the basis of NMR ontogeny in social insects. Thus, to investigate the mechanisms underlying the ontogeny of NMR in Polistes wasps, a model genus in recognition studies, and their different role in determining recognition abilities, we subjected Polistes dominula workers to different olfactory experiences in different phases of their life before inserting them into the social environment of a novel colony and testing them in recognition bioassays. Our results show that workers develop their NMR abilities based on their social context rather than through pre-imaginal and early learning or self-referencing. Our study demonstrates that the social context represents the major component shaping recognition abilities in a social wasp, therefore shedding new light on the ontogeny of recognition in paper wasps and prompting the reader to rethink about the traditional knowledge at the basis of the recognition in social insects. This article is part of the theme issue 'Signal detection theory in recognition systems: from evolving models to experimental tests'.

Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection.

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade-offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic-resistant Gram-negative bacteria Escherichia coli XL1-Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS-CAO1 and Heterorhabditis bacteriophora ItH-LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.

Saccharomyces cerevisiae Induces Immune Enhancing and Shapes Gut Microbiota in Social Wasps.

Trained immunity is the enhanced response of the innate immune system to a secondary infection after an initial encounter with a microorganism. This non-specific response to reinfection is a primitive form of adaptation that has been shown to be conserved from plants to mammals. Insects lack an acquired immune component and rely solely on an innate one, and they have expanded it upon traits of plasticity and adaptation against pathogens in the form of immune priming. The recent discoveries of the role of Saccharomyces cerevisiae in the insect's ecology and the ability of this yeast to induce trained immunity in different organisms suggest that insects could have developed mechanisms of adaptation and immune enhancing. Here, we report that two yeast strains of S. cerevisiae, previously shown to induce trained immunity in mammals, enhance resistance to Escherichia coli infection in the paper wasp Polistes dominula. The reduction of injected E. coli load by S. cerevisiae strains was statistically significant in future foundresses but not in workers, and this occurs before and after hibernation. We thus investigated if the effect on E. coli was mirrored by variation in the gut microbiota composition. Foundresses, showing immune enhancing, had statistically significant changes in composition and diversity of gut bacterial communities but not in the fungal communities. Our results demonstrate that S. cerevisiae can prime insect responses against bacterial infections, providing an advantage to future foundress wasps to carry these microorganisms. Understanding the mechanisms involved in the generation of specific and long-lasting immune response against pathogenic infections in insects and the influence of the induction of trained immunity on the commensal gut microbiota could have a major impact on modern immunology.

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.

Natural biocide disrupts nestmate recognition in honeybees.

Honeybee colonies are under the threat of many stressors, biotic and abiotic factors that strongly affect their survival. Recently, great attention has been directed at chemical pesticides, including their effects at sub-lethal doses on bee behaviour and colony success; whereas the potential side effects of natural biocides largely used in agriculture, such as entomopathogenic fungi, have received only marginal attention. Here, we report the impact of the fungus Beauveria bassiana on honeybee nestmate recognition ability, a crucial feature at the basis of colony integrity. We performed both behavioural assays by recording bee guards' response towards foragers (nestmate or non-nestmate) either exposed to B. bassiana or unexposed presented at the hive entrance, and GC-MS analyses of the cuticular hydrocarbons (CHCs) of fungus-exposed versus unexposed bees. Our results demonstrated that exposed bees have altered cuticular hydrocarbons and are more easily accepted into foreign colonies than controls. Since CHCs are the main recognition cues in social insects, changes in their composition appear to affect nestmate recognition ability at the colony level. The acceptance of chemically unrecognizable fungus-exposed foragers could therefore favour forager drift and disease spread across colonies.

Preference of Polistes dominula wasps for trumpet creepers when infected by Xenos vesparum: A novel example of co-evolved traits between host and parasite.

The parasitic insect Xenos vesparum induces noticeable behavioral and physiological changes-e.g. castration-in its female host, the paper wasp Polistes dominula: parasitized putative workers avoid any colony task and desert the colony to survive in the nearby vegetation, like future queens and males do. In this long-term observational study, we describe the spectacular attraction of parasitized workers towards trumpet creeper bushes (Campsis radicans) in early-summer. Two thirds of all wasps that we sampled on these bushes were parasitized, whereas the parasite prevalence was much lower in our study area and most wasps sampled on other nearby flowering bushes were non-parasitized. First, we describe the occurrence and consistency of this phenomenon across different sites and years. Second, we evaluate the spatial behavior of parasitized wasps on C. radicans bushes, which includes site-fidelity, exploitation and defense of rich extra-floral nectaries on buds and calices. Third, we record two critical steps of the lifecycle of X. vesparum on C. radicans: the parasite's mating and a summer release of parasitic larvae, that can infect larval stages of the host if transported to the host's nest. In a nutshell, C. radicans bushes provide many benefits both to the parasite X. vesparum and to its host: they facilitate the parasite's mating and bivoltine lifecycle, a phenomenon never described before for this parasite, while, at the same time, they provide the wasp host with shelter inside trumpet flowers and extrafloral gland secretions, thus likely enhancing host survival and making it a suitable vector for the infection.

Antennal Protein Profile in Honeybees: Caste and Task Matter More Than Age.

Reproductive and task partitioning in large colonies of social insects suggest that colony members belonging to different castes or performing different tasks during their life (polyethism) may produce specific semiochemicals and be differently sensitive to the variety of pheromones involved in intraspecific chemical communication. The main peripheral olfactory organs are the antennal chemosensilla, where the early olfactory processes take place. At this stage, members of two different families of soluble chemosensory proteins [odorant-binding proteins (OBPs) and chemosensory proteins (CSPs)] show a remarkable affinity for different odorants and act as carriers while a further family, the Niemann-Pick type C2 proteins (NPC2) may have a similar function, although this has not been fully demonstrated. Sensillar lymph also contains Odorant degrading enzymes (ODEs) which are involved in inactivation through degradation of the chemical signals, once the message is conveyed. Despite their importance in chemical communication, little is known about how proteins involved in peripheral olfaction and, more generally antennal proteins, differ in honeybees of different caste, task and age. Here, we investigate for the first time, using a shotgun proteomic approach, the antennal profile of honeybees of different castes (queens and workers) and workers performing different tasks (nurses, guards, and foragers) by controlling for the potential confounding effect of age. Regarding olfactory proteins, major differences were observed between queens and workers, some of which were found to be more abundant in queens (OBP3, OBP18, and NPC2-1) and others to be more abundant in workers (OBP15, OBP21, CSP1, and CSP3); while between workers performing different tasks, OBP14 was more abundant in nurses with respect to guards and foragers. Apart from proteins involved in olfaction, we have found that the antennal proteomes are mainly characterized by castes and tasks, while age has no effect on antennal protein profile. Among the main differences, the strong decrease in vitellogenins found in guards and foragers is not associated with age.

The trap of sex in social insects: from the female to the male perspective.

The phenotype of male Hymenoptera and the peculiar role of males has been neglected and greatly understudied, given the spectacular cooperative behavior of female social insects. In social insects there has been considerable progress in understanding the molecular mechanisms behind haplodiploid sex determination but, beyond that, very little is known concerning the neural, endocrine, and genetic correlates of sexual selection in males. An opportunity is being missed: the male phenotype in Hymenoptera is a natural experiment to compare the drives of natural versus sexual selection. In contrast to females, males do not work, they usually display far from the nest to gain mates, compete among rivals in nuptial flights or for a symbolic territory at leks, and engage in direct or ritualized conflicts. By comparing the available data on male paper wasps with studies on other social Hymenoptera, we summarize what we currently know about the physical, hormonal, neural and behavioral traits in a model system appropriate to examine current paradigms on sexual selection. Here we review male behavior in social Hymenoptera beyond sex stereotypes: the subtle role of "drones" in the colony, the lack of armaments and ornaments, the explosive mating crowds, the "endurance" race, the cognitive bases of the "choosy" male and his immune defense. Social insect males are not just simple-minded mating machines, they are shaped, constrained and perhaps trapped by sexual selection.

Novel insights into the ontogeny of nestmate recognition in Polistes social wasps.

The importance of early experience in animals' life is unquestionable, and imprinting-like phenomena may shape important aspects of behaviour. Early learning typically occurs during a sensitive period, which restricts crucial processes of information storage to a specific developmental phase. The characteristics of the sensitive period have been largely investigated in vertebrates, because of their complexity and plasticity, both in behaviour and neurophysiology, but early learning occurs also in invertebrates. In social insects, early learning appears to influence important social behaviours such as nestmate recognition. Yet, the mechanisms underlying recognition systems are not fully understood. It is currently believed that Polistes social wasps are able to discriminate nestmates from non-nestmates following the perception of olfactory cues present on the paper of their nest, which are learned during a strict sensitive period, immediately after emergence. Here, through differential odour experience experiments, we show that workers of Polistes dominula develop correct nestmate recognition abilities soon after emergence even in absence of what have been so far considered the necessary cues (the chemicals spread on nest paper). P. dominula workers were exposed for the first four days of adult life to paper fragments from their nest, or from a foreign conspecific nest or to a neutral condition. Wasps were then transferred to their original nests where recognition abilities were tested. Our results show that wasps do not alter their recognition ability if exposed only to nest material, or in absence of nest material, during the early phase of adult life. It thus appears that the nest paper is not used as a source of recognition cues to be learned in a specific time window, although we discuss possible alternative explanations. Our study provides a novel perspective for the study of the ontogeny of nestmate recognition in Polistes wasps and in other social insects.