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Biopesticides and insect pollinators: Detrimental effects, outdated guidelines, and future directions.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

Differential investment in visual and olfactory brain regions is linked to the sensory needs of a wasp social parasite and its host.

Obligate insect social parasites evolve traits to effectively locate and then exploit their hosts, whereas hosts have complex social behavioral repertoires, which include sensory recognition to reject potential conspecific intruders and heterospecific parasites. While social parasites and host behaviors have been studied extensively, less is known about how their sensory systems function to meet their specific selective pressures. Here, we compare investment in visual and olfactory brain regions in the paper wasp Polistes dominula, and its obligate social parasite P. sulcifer, to explore the links among sensory systems,brain and behavior. Our results show significant relative volumetric differences between these two closely related species, consistent with their very different life histories. Social parasites show proportionally larger optic lobes and central complex to likely navigate long-distance migrations and unfamiliar landscapes to locate the specific species of hosts they usurp. Contrastingly, hosts have larger antennal lobes and calyces of the mushroom bodies compared with social parasites, as predicted by their sensory means to maintain social cohesion via olfactory signals, allocate colony tasks, forage, and recognize conspecific and heterospecific intruders. Our work suggests how this tradeoff between visual and olfactory brain regions may facilitate different sensory adaptations needed to perform social and foraging tasks by the host, including recognition of parasites, or to fly long distances and successful host localizing by the social parasite.

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.

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.

Inquiline social parasites as tools to unlock the secrets of insect sociality.

Insect societies play a crucial role in the functioning of most ecosystems and have fascinated both scientists and the lay public for centuries. Despite the long history of study, we are still far from understanding how insect societies have evolved and how social cohesion in their colonies is maintained. Here we suggest inquiline social parasites of insect societies as an under-exploited experimental tool for understanding sociality. We draw on examples from obligate inquiline (permanent) social parasites in wasps, ants and bees to illustrate how these parasites may allow us to better understand societies and learn more about the evolution and functioning of insect societies. We highlight three main features of these social parasite-host systems-namely, close phylogenetic relationships, strong selective pressures arising from coevolution and multiple independent origins-that make inquiline social parasites particularly suited for this aim; we propose a conceptual comparative framework that considers trait losses, gains and modifications in social parasite-host systems. We give examples of how this framework can reveal the more elusive secrets of sociality by focusing on two cornerstones of sociality: communication and reproductive division of labour. Together with social parasites in other taxonomic groups, such as cuckoos in birds, social parasitism has a great potential to reveal the mechanisms and evolution of complex social groups. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

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.

Adult-larval vibrational communication in paper wasps: the role of abdominal wagging in Polistes dominula.

Communication through vibrational signals is widespread among social insects and regulates crucial social activities. Females of the social wasp Polistes dominula produce substrate-borne vibrations on the combs by performing a conspicuous abdominal oscillatory behavior, known as abdominal wagging. Several studies have reported correlative evidence in support of its signaling role, but direct evidence is still lacking. Because abdominal wagging is strictly associated with the presence of larvae in the nest and with cell inspection, it has been suggested that it could be involved in adult-larvae communication. According to this hypothesis, abdominal wagging vibrations would have short-term effects related to food and trophallactic exchanges between adults and larvae by modulating salivary secretion (decreasing its amount, to prepare larvae to receive food, or stimulating the release of larval saliva to adults). Here, by using an electro-magnetic shaker, we assessed, for the first time, the short-term effects of abdominal wagging on larval behavior by recording larval responses and by measuring the amount of saliva released immediately after abdominal wagging playback. Our results show that larvae are able to perceive the substrate-borne vibrations produced by abdominal wagging and react by increasing the movement of their body, possibly in order to attract the attention of adult females during feeding nest inspection. Yet, we found that vibrations neither increase nor decrease the release of larval saliva. Our results support the hypothesis of the alleged role of vibrations in adult-larvae communications; however, they do not support the long-lasting hypothesis of salivary release modulation.

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.

Social parasitism and the molecular basis of phenotypic evolution.

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer-Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization.

Effects of the diet on the microbiota of the red palm weevil (Coleoptera: Dryophthoridae).

Rhynchophorus ferrugineus, also known as the red palm weevil, is regarded as the major pest of palm trees. Although studies of the microbiota associated with this species have been performed in recent years, little attention has been dedicated to the influence of the diet in shaping the host bacterial community. Here, we investigated the influence of food sources (i.e. palm tissues vs apple based substrate) on the microbial diversity associated with RPW, which was compared with the microbiota associated with wild individuals of the sister species Rhynchophorus vulneratus. The bacterial characterization was performed using a culture independent approach, i.e. the 16S rRNA pyrotag, and a culture dependent approach for a subset of the samples, in order to obtain bacterial isolates from RPW tissues. The bacterial community appeared significantly influenced by diet. Proteobacteria resulted to be the most abundant clade and was present in all the specimens of the three examined weevil groups. Within Proteobacteria, Enterobacteriaceae were identified in all the organs analysed, including hemolymph and reproductive organs. The apple-fed RPWs and the wild R. vulneratus showed a second dominant taxon within Firmicutes that was scarcely present in the microbiota associated with palm-fed RPWs. A comparative analysis on the bacteria associated with the palm tissues highlighted that 12 bacterial genera out of the 13 identified in the plant tissues were also present in weevils, thus indicating that palm tissues may present a source for bacterial acquisition.

Facial markings in the social cuckoo wasp Polistes sulcifer: No support for the visual deception and the assessment hypotheses.

Insect social parasites have to conquer a host colony by overcoming its defensive barriers. In addition to increased fighting abilities, many social parasites evolved sophisticated sensory deception mechanisms to elude host colonies defenses by exploiting host communication channels. Recently, it has been shown that the conspicuous facial markings of a paper wasp social parasite, Polistes sulcifer, decrease the aggressiveness of host foundresses. Two main hypotheses stand as explanations of this phenomenon: visual sensory deception (i.e. the black patterning reduces host aggression by exploiting the host visual communication system) and visual quality assessment (i.e. facial markings reduce aggressiveness as they signal the increased fighting ability of parasites). Through behavioral assays and morphological measurements we tested three predictions resulting from these hypotheses and found no support either for the visual sensory deception or for the quality assessment to explain the reduction in host aggressiveness towards the parasite. Our results suggest that other discrimination processes may explain the observed phenomenon.

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.

Sensing the intruder: a quantitative threshold for recognition cues perception in honeybees.

The ability to discriminate among nestmates and non-nestmate is essential to defend social insect colonies from intruders. Over the years, nestmate recognition has been extensively studied in the honeybee Apis mellifera; nevertheless, the quantitative perceptual aspects at the basis of the recognition system represent an unexplored subject in this species. To test the existence of a cuticular hydrocarbons' quantitative perception threshold for nestmate recognition cues, we conducted behavioural assays by presenting different amounts of a foreign forager's chemical profile to honeybees at the entrance of their colonies. We found an increase in the explorative and aggressive responses as the amount of cues increased based on a threshold mechanism, highlighting the importance of the quantitative perceptual features for the recognition processes in A. mellifera.