The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

O comportamento social pode ser descrito como qualquer atividade de interação intraespecífica incluindo a escolha entre parceiros reprodutivos, reconhecimento da espécie, comportamento altruísta e organização da sociedade animal. Entre as espécies de animais mais sintonizadas com seu ambiente social estão os insetos que, como por exemplo nas espécies de abelhas das tribos Apini e Meliponini, apresentam um padrão complexo de socialidade conhecido como comportamento altamente eussocial. As abelhas constituem um grupo ideal para o estudo das bases da evolução deste comportamento, pois apresentam uma grande diversidade de organização social, desde espécies solitárias até altamente eussociais. Embora a evolução da eussocialidade tenha sido motivo de muitos estudos, as mudanças genéticas envolvidas nesse processo não são completamente conhecidas. Dados da literatura fornecem um ponto de partida para o entendimento da relação entre alterações gênicas específicas e a eussocialidade, mas questões fundamentais na evolução do comportamento social ainda precisam ser respondidas. Recentemente, novas tecnologias de sequenciamento têm permitido o estudo de organismos modelo e não modelo de forma mais detalhada e não direcional. Análises deste tipo são promissoras para o estudo evolutivo de características complexas como o comportamento. Neste contexto, realizamos um amplo estudo sobre as bases moleculares envolvidas em diferentes características comportamentais relacionadas à evolução da socialidade em abelhas. Para tanto, o padrão global de expressão de genes, em espécies e fases do desenvolvimento distintas, foram analisados comparativamente através de múltiplas abordagens. No Capítulo 1, utilizamos contaminantes do transcriptoma da abelha solitária Tetrapedia diversipes para analisar os recursos florais utilizados por esta espécie em suas duas gerações reprodutivas. Neste estudo concluímos que a riqueza de espécies visitadas durante a primeira geração é muito maior do que durante a segunda geração, o que está provavelmente relacionado à floração de primavera durante o primeiro período reprodutivo. No Capítulo 2, verificamos que o padrão de expressão dos genes das fêmeas fundadoras possivelmente afeta o desenvolvimento larval em T. diversipes. O padrão bivoltino de reprodução desta espécie, com diapausa em uma das gerações, pode ser importante para a evolução do comportamento social. Além disso, entre os genes possivelmente envolvidos nessa característica, podemos encontrar genes mitocondriais e lncRNAs. Os resultados obtidos no Capítulo 3 sugerem que a especialização em subcastas de operárias ocorreu posteriormente nas diferentes linhagens de abelhas, envolvendo genes específicos. No entanto, esses genes afetam processos biológicos comuns nas diferentes espécies. Por sua vez, o Capítulo 4 apresenta um método promissor para a identificação de genes comportamentais em diferentes espécies de abelhas, através de uma análise de expressão comparativa. Com base nessas análises, 787 genes comportamentais, que possivelmente fazem parte de um toolkit eussocial em abelhas, foram encontrados. O padrão de metilação desses genes, em espécies com diferentes níveis sociais, indicou ainda que o contexto genômico da metilação pode ser relevante para eussocialidade. Os resultados obtidos nesses estudos apresentam novas perspectivas metodológicas e evolutivas para o estudo da evolução do comportamento social em abelhas / The social behaviour can be widely described as any intraspecific interaction in the animal life, including but not restricted to, female choice, species recognition, altruistic behaviour and the organization of animal society. Among the animal species most attuned to their social environment are the insects that, for example, in the Apini and Meliponini tribes, present a complex behaviour known as highly eusocial. Bees are an ideal group to study the evolution of the social behaviour because they have a great diversity of social life styles that evolved independently. The tribes Apini and Meliponini comprise only highly eusocial species whereas various levels of sociality can be detected in other tribes, being most bees indeed solitary. Although the evolution of eusociality has been the subject of many studies, the genetic changes involved in the process have not been completely understood. Results from studies conducted so far provide a starting point for the connection between specific genetic alterations and the evolution of eusocial behaviour. However fundamental questions about this process are still open. Recently, new sequencing technologies have allowed genetic studies of model and non-model organisms in a deep and non-directional way, which is promising for the study of complex characteristics. Herein, we present a broad analysis of the molecular bases of different behavioural characteristics related to the evolution of sociality in bees. To that end, the global expression pattern of genes involved in different behavioural features, in a number of bee species and distinct developmental stages, was comparatively studied using multiple approaches. Through these approaches different results were obtained. In Chapter 1, we used contaminant transcripts from the solitary bee Tetrapedia diversipes to identify the plants visited by this bee, during its two reproductive generations. These contaminant transcripts revealed that the richness of plant species visited during the first reproductive generation was considerably greater than during the second generation. Which is probably related to the floral boom occurring in spring during the first reproductive period. In Chapter 2, data suggests that the expression pattern in foundresses affect larval development in T. diversipes. The bivoltinism presented by this species, with diapause in one generation, might be an important feature for the evolution of sociality. Our results suggest that mitochondrial genes and lncRNAs are involved in this reproductive pattern. Results described in Chapter 3 indicate that specialization in worker subcastes occurred posteriorly in distinct bee lineages, driven by specific genes. However, these genes affected common biological processes in the different species. In Chapter 4 is described a promising analyses method to identify, comparatively, genes involved in bee social behaviour. Using this approach, we identified 787 behavioural genes that might be involved in social behaviour of different species. The methylation pattern of these genes suggests that the DNA context in which methylation marks occur, might be especially relevant to bee sociality. Results obtained here presents new methodological and evolutionary approaches to the study of social behaviour in bees

Social insects are ecologically dominant because of their specialized, cooperative castes. Reproductive queens lay eggs, while workers take part in brood rearing, nest defense, and foraging. These cooperative castes are a prime example of phenotypic plasticity, whereby a single genetic code gives rise to variation in form and function based on environmental differences. Thus, social insects are well suited for studying mechanisms which give rise to and maintain phenotypic plasticity. At the molecular level, phenotypic plasticity coincides with the differential expression of genes. This dissertation examines the molecular evolution of genes with differential expression between discrete phenotypic or environmental contexts, represented chiefly by female queen and worker castes in social insects. The studies included herein examine evolution at three important levels of biological information: (i) gene expression, (ii) modifications to DNA in the form of methylation, and (iii) protein-coding sequence. From these analyses, a common theme has emerged: genes with differential expression among castes frequently exhibit signatures of relaxed selective constraint relative to ubiquitously expressed genes. Thus, genes associated with phenotypic plasticity paradoxically exhibit modest importance to overall fitness but exceptional evolutionary potential, as illustrated by the success of the social insects.

The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

The evolution of nonreproductive castes is a defining characteristic of eusociality. The function and developmental regulation of the altruistic worker and soldier caste is the central element contributing to major advantages of eusociality over solitary animals. The soldier caste is the first evolved sterile caste in termites. Their primary function is believed to be colony defense. However, the function and development of termite soldiers remains largely unknown. Because of their apparent morphological adaptation for fighting and their limited behavior repertoire, our understanding of colony defense by termite soldiers is limited to their physical defense. In addition, we know little about the molecular mechanisms mediating soldier development. In Chapters 2 and 3 I discuss the role of the soldier caste under competition risk. By exposing the Eastern subterranean termite Reticulitermes flavipes to cues of a competitor termite species, I found that exposure to competitor cues reduced feeding, compromised growth and survival of R. flavipes workers. The presence of R. flavipes soldiers largely ameliorated these negative impacts. At the transcriptional level, R. flavipes soldiers can counteract the effects of competitor cues on worker head gene expression. This counteracting effect seems to be associated with genes in metabolism and immunity. These studies demonstrate that competition can affect a termite colony’s fitness by either competitors physically invading the colony and causing damage or cues from competitors inducing a stress response in termite colony members. More importantly, soldiers can contribute to colony fitness by physically engaging in combat, but also by enhancing colony members’ survival under competitor-cue exposure. In Chapter 4, I describe the molecular mechanism mediating soldier-caste differentiation. I cloned the full length cDNA sequence of the R. flavipes Methoprene-tolerance (Met) gene, a gene encoding a putative receptor for juvenile hormones. Using RNA interference, I studied the function of Met and found that this gene essentially mediates the JH-dependent soldier-caste differentiation in termites.

Soldier caste is the oldest altruistic caste in termites, highly specialized for defensive tasks. Therefore, a number of adaptations in anatomy and behaviour can be observed in soldier, lacking in other termite castes. The aim of this study was to verify the hypothesis that in disturbed groups the soldiers will prefer to stand by and defend the reproductives, the most valuable individuals. Therefore, I studied in two species of the genus Prorhinotermes (P. simplex and P. canalifrons) the ability of soldiers to discriminate neotenic reproductives and provide them with defence. In agreement with the initial hypothesis, the soldiers of both species prooved to be able of an instantaneous recognition of reproductives and remained close to them. In P. simplex, the neotenic female was the most attractive individual, while in P. canalifrons, neotenics of both sexes were equally attractive. The observed preference of reproductives appears to occur only with a certain age of the reproductives in incipient colonies, along with their sexual maturation. Caste specificity of cuticular lipids supports the presumption that the caste recognition is based on olphactoric cues in the cuticle of reproductives.

Bees are aculeate hymenopterans, which, altogether, comprise approximately 20,000 valid species divided into seven families, among which Apidae is one of the best known groups. In addition to the species richness, a remarkable diversity of social behaviors is known to exist within this group, ranging from strict solitary taxa (over 80% of bee species) to groups showing highly sophisticated behaviors related to life in society. One of the most fascinating groups of bees in this respect is that of corbiculate bees (Apidae: Apinae: Apini). Amongst its four subtribes, Apina and Meliponina comprise the so-called \"fixed-caste eusocial\" groups. Phylogenetic relationships of corbiculate bees have been under intense controversy for the last two decades, thus hampering a robust understanding of the evolution of eusocial traits. Most hypotheses based on morphological/behavioral data point to a clade uniting Apina + Meliponina, thus favoring a single origin scenario for the evolution of fixed-caste eusociality. Molecular datasets, on the other hand, consistently indicated independent origins for this cluster of traits. In this contribution, the main goal was to re-evaluate the phylogenetic relationships between the major lineages of corbiculate bees exploring new sources of information from internal skeletal structures of exoskeleton. It were provided descriptions/discussions on some important morphological complexes of the head capsule, mandibles, sitophore, propectus, meso/metafurca and mesophragma of bees. Also, it was attempted to standardize the current morphological terminology used for bees to that used for other Hymenoptera. A set of different phylogenetic analysis with the main lineages of Apidae emphasizing the corbiculate clade was conducted. The complete morphological matrix with 93 characters from external morphology and 42 from internal structures of exoskeleton resulted into two most parsimonious trees with 376 steps, CI=45 and RI=78, which were congruent with the current morphological/behavioral scenario for the corbiculate relationships: Euglossina + (Bombina + (Apina + Meliponina)). It was demonstrated that characters extracted from internal structures of exoskeleton, although poorly explored, can provide critical evidence to elucidate the corbiculate controversy. Moreover, despite the inclusion of new sources of data is fundamental to elucidate this issue, some efforts should be made to reevaluate many characters currently in (re)use. Also, the inclusion of fossil information and use of new technologies for phenotypic studies can open new windows for future comparative morphological studies in bees / Abelhas são himenópteros aculeados que, ao todo, compreendem aproximadamente 20.000 espécies válidas distribuídas em sete famílias, das quais Apidae é um dos grupos mais conhecidos. Além da diversidade de espécies, uma notável diversidade de comportamentos sociais é conhecida para esse grupo, desde táxons estritamente solitários (mais de 80% das espécies de abelhas) a grupos exibindo comportamentos altamente sofisticados relacionados à vida em sociedade. Um dos grupos mais fascinantes nesse aspecto é o das abelhas corbiculadas (Apidae: Apinae: Apini). Dentre suas quatro subtribos, Apina e Meliponina compreendem os chamados grupos eussociais de casta-fixa. As relações filogenéticas entre abelhas corbiculadas têm estado sob intensa controvérsia nas duas últimas décadas, o que dificulta a compreensão robusta da evolução dos atributos da eussocialidade. A maioria das hipóteses baseadas em dados morfológicos/comportamentais apontam para um clado unindo Apina + Meliponina, portanto favorecendo um cenário de origem única para a eusocialidade de casta-fixa. Conjuntos de dados moleculares, por outro lado, indicam consistentemente origens independentes para esse conjunto de atributos. Nesta contribuição, o objetivo principal foi reavaliar as relações filogenéticas entre as principais linhagens de abelhas corbiculadas, explorando novas fontes de informação das estruturas internas do exoesqueleto. Foram fornecidas descrições/discussões sobre alguns importantes complexos morfológicos da cabeça, mandíbulas, sitóforo, meso/metafurca e mesofragma de abelhas. Além disso, buscou-se padronizar a atual terminologia morfológica usada para abelhas com aquela usada para outros Hymenoptera. Um conjunto de diferentes análises com as principais linhagens de Apidae, enfatizando o clado das corbiculadas, foi conduzido. A matrix morfológica completa com 93 caracteres de morfologia externa e 42 de estruturas internas do exoesqueleto resultou em duas árvores mais parcimoniosas com 376 passos, CI=45 e RI=78, que são congruentes com o cenário morfológico/comportamental atual para as relações filogenéticas das corbiculadas: Euglossina + (Bombina + (Apina + Meliponina)). Foi demonstrado que caracteres de estruturas internas do exoesqueleto, apesar de pouco explorados, podem fornecer evidências críticas para elucidar a controvérsia das corbiculadas. Ademais, apesar da inclusão de novas fontes de dados ser fundamental para essa questao, algum esforço deve ser feito para se reavaliar muitos caracteres atualmente em (re)uso. Além disso, a inclusão de informação de fósseis e uso de novas tecnologias para estudos fenotípicos podem abrir novas janelas para futuros estudos morfológicos em abelhas

Bees are aculeate hymenopterans, which, altogether, comprise approximately 20,000 valid species divided into seven families, among which Apidae is one of the best known groups. In addition to the species richness, a remarkable diversity of social behaviors is known to exist within this group, ranging from strict solitary taxa (over 80% of bee species) to groups showing highly sophisticated behaviors related to life in society. One of the most fascinating groups of bees in this respect is that of corbiculate bees (Apidae: Apinae: Apini). Amongst its four subtribes, Apina and Meliponina comprise the so-called \"fixed-caste eusocial\" groups. Phylogenetic relationships of corbiculate bees have been under intense controversy for the last two decades, thus hampering a robust understanding of the evolution of eusocial traits. Most hypotheses based on morphological/behavioral data point to a clade uniting Apina + Meliponina, thus favoring a single origin scenario for the evolution of fixed-caste eusociality. Molecular datasets, on the other hand, consistently indicated independent origins for this cluster of traits. In this contribution, the main goal was to re-evaluate the phylogenetic relationships between the major lineages of corbiculate bees exploring new sources of information from internal skeletal structures of exoskeleton. It were provided descriptions/discussions on some important morphological complexes of the head capsule, mandibles, sitophore, propectus, meso/metafurca and mesophragma of bees. Also, it was attempted to standardize the current morphological terminology used for bees to that used for other Hymenoptera. A set of different phylogenetic analysis with the main lineages of Apidae emphasizing the corbiculate clade was conducted. The complete morphological matrix with 93 characters from external morphology and 42 from internal structures of exoskeleton resulted into two most parsimonious trees with 376 steps, CI=45 and RI=78, which were congruent with the current morphological/behavioral scenario for the corbiculate relationships: Euglossina + (Bombina + (Apina + Meliponina)). It was demonstrated that characters extracted from internal structures of exoskeleton, although poorly explored, can provide critical evidence to elucidate the corbiculate controversy. Moreover, despite the inclusion of new sources of data is fundamental to elucidate this issue, some efforts should be made to reevaluate many characters currently in (re)use. Also, the inclusion of fossil information and use of new technologies for phenotypic studies can open new windows for future comparative morphological studies in bees / Abelhas são himenópteros aculeados que, ao todo, compreendem aproximadamente 20.000 espécies válidas distribuídas em sete famílias, das quais Apidae é um dos grupos mais conhecidos. Além da diversidade de espécies, uma notável diversidade de comportamentos sociais é conhecida para esse grupo, desde táxons estritamente solitários (mais de 80% das espécies de abelhas) a grupos exibindo comportamentos altamente sofisticados relacionados à vida em sociedade. Um dos grupos mais fascinantes nesse aspecto é o das abelhas corbiculadas (Apidae: Apinae: Apini). Dentre suas quatro subtribos, Apina e Meliponina compreendem os chamados grupos eussociais de casta-fixa. As relações filogenéticas entre abelhas corbiculadas têm estado sob intensa controvérsia nas duas últimas décadas, o que dificulta a compreensão robusta da evolução dos atributos da eussocialidade. A maioria das hipóteses baseadas em dados morfológicos/comportamentais apontam para um clado unindo Apina + Meliponina, portanto favorecendo um cenário de origem única para a eusocialidade de casta-fixa. Conjuntos de dados moleculares, por outro lado, indicam consistentemente origens independentes para esse conjunto de atributos. Nesta contribuição, o objetivo principal foi reavaliar as relações filogenéticas entre as principais linhagens de abelhas corbiculadas, explorando novas fontes de informação das estruturas internas do exoesqueleto. Foram fornecidas descrições/discussões sobre alguns importantes complexos morfológicos da cabeça, mandíbulas, sitóforo, meso/metafurca e mesofragma de abelhas. Além disso, buscou-se padronizar a atual terminologia morfológica usada para abelhas com aquela usada para outros Hymenoptera. Um conjunto de diferentes análises com as principais linhagens de Apidae, enfatizando o clado das corbiculadas, foi conduzido. A matrix morfológica completa com 93 caracteres de morfologia externa e 42 de estruturas internas do exoesqueleto resultou em duas árvores mais parcimoniosas com 376 passos, CI=45 e RI=78, que são congruentes com o cenário morfológico/comportamental atual para as relações filogenéticas das corbiculadas: Euglossina + (Bombina + (Apina + Meliponina)). Foi demonstrado que caracteres de estruturas internas do exoesqueleto, apesar de pouco explorados, podem fornecer evidências críticas para elucidar a controvérsia das corbiculadas. Ademais, apesar da inclusão de novas fontes de dados ser fundamental para essa questao, algum esforço deve ser feito para se reavaliar muitos caracteres atualmente em (re)uso. Além disso, a inclusão de informação de fósseis e uso de novas tecnologias para estudos fenotípicos podem abrir novas janelas para futuros estudos morfológicos em abelhas

The social mole-rats have often been typecast as extreme examples of mammalian sociality. With their pronounced reproductive skew, status-related contrasts in lifespan and morphology, and the suggestion of a division of labour amongst helpers, mole-rat societies have repeatedly been likened to the structurally complex societies of some eusocial insects. However, because few studies of mole-rats have quantified individual variation in growth and behaviour across long periods of development, it has remained unclear the extent to which mole-rat societies, and the features of individuals within them, should be considered unique amongst social vertebrates. In this thesis, I examine life history variation in Damaraland mole-rats Fukomys damarensis from three perspectives- growth, behaviour, and ageing- to explore how individual developmental trajectories contribute to, and are influenced by, the structure of mole-rat societies. First, I use a large longitudinal dataset to test for the presence of behavioural specialisation in non-breeding mole-rat helpers. I find no indication of individual specialisation in cooperative activities. Instead, individual differences in helping behaviour are largely the result of age-related changes in the extent to which individuals commit to all forms of helping (Chapter 3); refuting the notion of helper castes. I then focus on the variation in growth across non-breeders, developing a novel biphasic model to accurately quantify sex differences in growth and explore the influence of social effects on growth trajectories (Chapter 4). Despite the proposition of intense intrasexual competition in mole-rat societies, there was no clear signature of sex-specific competition on helper growth trajectories. A more conspicuous form of socially-mediated growth in mole-rats is the secondary growth spurt displayed by females that have acquired the dominant breeding position, causing them to become larger and more elongated. By experimentally controlling reproduction in age-matched siblings, I show that rather than being stimulated by the removal from reproductive suppression, this adaptive morphological divergence is achieved through a lengthening of the lumbar vertebrae when breeding is commenced (Chapter 5). With contrasts in size and shape following the acquisition of the breeding role, this status-related growth pattern shares similarities with growth in naked mole-rats and other social vertebrates. Breeders also show a twofold greater lifespan than non-breeders in Fukomys mole-rats, prompting the suggestion that the transition to dominance also sets individuals onto a slower ageing trajectory. To date, there is little evidence to support a physiological basis to lifespan extension in breeders. This assertion is bolstered by the absence of longer telomeres or slower rates of telomere attrition in breeding females compared to non-breeding females residing in groups (Chapter 6), each of which might be expected if breeders age more slowly. I argue that previous studies exploring status-related ageing in captive Fukomys mole-rats have overlooked the importance of demographic processes (and associated behavioural influences) on mortality schedules. Irrespective of the proximate basis of the longer lifespan of breeders, at an interspecific level the social mole-rats are unusually long-lived for their size. A recent large-scale comparative analysis concluded that prolonged lifespan is a general characteristic of all mammalian cooperative breeders, but this conclusion is premature, as in most of the major clades containing both cooperative and non-cooperative species there is no consistent trend towards lifespan extension in cooperative species (Chapter 7). In the case of mole-rats, it seems more likely that their exceptional longevity arises principally from their subterranean habits and related reductions in extrinsic mortality. Overall, these findings demonstrate that cooperative breeding has important consequences for individual life histories, but there is no strong basis for the claim that Damaraland mole-rat societies are markedly different in form than other cooperative breeding societies.

This thesis has looked at the proximate mechanisms by which eusociality is maintained in colonies of the primitively eusocial wasp Ropalidia marginata. Unlike other typical primitively eusocial species, the R, marginata queen is remarkably docile and non-interactive and hence cannot possibly use aggression to maintain her status. Recent evidence hints at pheromonal queen signalling through the Dufour’s gland. Hence, queen-worker difference in Dufour’s gland composition has been studied in details. Queens and workers differ with respect to overall composition of Dufour’s glands, categories of compounds, and individual compounds as well. The Dufour’s gland compounds may be having a bouquet effect in queen signalling, with individual compounds being less important than the overall composition. The queen pheromone also appears to be an honest signal of fertility, as compounds that differ consistently between queens and workers are correlated with ovarian development of queens, and solitary foundresses and potential queens, who are intermediate between queens and workers in ovarian development, are intermediate in their Dufour’s gland profile as well. When the queen is removed from a colony, one of the workers (potential queen, PQ) shows high aggression, and if the queen is not returned, goes on to become the next queen of the colony. The aggression of PQ comes down as a function of time since queen removal, and correlated with this, the ovaries of PQ increase. Dufour’s gland profile of PQ is similar to workers immediately after queen removal, but comes closer to queens with passage of time. This hints at an interesting transition in maintenance of eusociality from “queen control” by aggression to “queen signal” by pheromone during the queen establishment phase. It has generally been assumed that one set of chemicals can carry multiple information, namely queen signal and colony signal. Initial statistical analysis of chemical composition data showed that perhaps both caste and colony signals can be conveyed by the Dufour’s gland compounds, but detailed analysis cast some doubt on this, as the Dufour’s gland compounds could not be separated into non-overlapping subsets with respect to importance in caste and colony discrimination. A bioassay showed that the wasps do not make colony discrimination from Dufour’s gland compounds. This suggests that the ability to statistically differentiate groups of organisms from their chemical profiles does not guarantee similar discrimination by the organisms themselves, emphasising the need for bioassays to resolve such issues.