Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Insect societies utilize advanced chemical communication systems to organize many aspects of their social life, which among others, include reproduction, thus maintaining colony homeostasis. The queen pheromone complex (QMP), dominated by (E)-9-keto-2-decenoic acid (9ODA) is of integral importance in regulating worker reproductive development. Unique characteristics, associated with reproductive dominance, enabled the successful establishment of Apis mellifera capensis workers as social parasites (or pseudoqueens) in colonies of the neighbouring A. m. scutellata. This suggested that producing a queenlike pheromonal bouquet is one of the proximate factors in their success. In this study we attempted to address the pheromone communication dilemma by investigating whether the phenotypic expression of mandibular gland signals in honeybee workers are under genetic and/or environmental influence. It was hypothesized that the mandibular gland profiles of queens and workers may be closely correlated to specific genotypes in the colony. However, different ageing and rearing environments (social context) can ultimately influence gene expression with respect to mandibular gland signals, highlighting the fact that environmental influences are not necessarily non-significant. In our experiments, both environmental/social conditions and genotypes of our test individuals were manipulated. The capensis workers used in our experiment from their native range (Western Cape area are refered to as native workers, while capensis parasitic workers, from the clonal parasitic lineage, were obtained from the Gauteng area. A. m. scutellata workers were obtained from their native range, north of the hybrid zone. Both native and parasitic workers showed the potential to become reproductively active, but the rapid pheromonal development of parasitic workers placed them at a reproductive advantage. Parasitic workers started producing low levels of 9HDA, the precursor to the queen substance 9ODA, between 12-24 hrs, while native workers only did so after 24 hrs. Despite this, rapid signal development did not culminate in the parasitic clones always pheromonally out-competing native workers. Withinin groups of native workers and a single clonal parasitic worker, the mandibular gland profiles of most workers were dominated by 9ODA and 9HDA (> 80% of extracts) with only 43% of the single parasitic workers producing higher amounts of 9ODA than native workers. Mandibular gland pheromone profiles converged in groups of workers sharing a greater proportion of genes, providing support for a link to genotypic affects. Workers that were 75 – 99% related diverged significantly from groups with lower levels of relatedness was largely due to the presences of 9ODA (Spearman’s rank correlation r = 0.66, p < 0.0001). Despite the tendency for signal to convergence in groups of closer relatedness a considerable amount of signal variability was also observed under varying social conditions. Workers originating from a single capensis queen but aged under queenright and queenless conditions had very distinct mandibular gland profiles (Wilks’ lambda λ = 0.118, χ2 = 331.002, p < 0.0001). This variability was thus a result of the social environment that the workers were exposed to. The physiological traits, namely mandibular gland pheromone production, linked to reproductive potential in honeybee workers seem to be determined by a combination of environmental and genetic factors. Queen mandibular gland pheromone biosynthesis is genetically predisposed in certain workers however the final oxidation step to 9ODA is strongly influenced by the social environment. The signal plasticity observed in this study is adaptive and assists workers to realize their reproductive potential. / AFRIKAANSE OPSOMMING: Insek gemeenskappe gebruik gevorderde chemiese kommunikasie sisteme om verskeie aspekte van sosiale lewe, onder andere reproduksie, te organiseer en sodoende word korf homeostasis handhaaf. Die feromoon kompleks van die koninginby, wat hoofsaaklik uit (E)-9-keto-2-decenoic acid (9ODA) bestaan speel ʼn belangrike rol in die regulering van reproduksie in heuningby werkers. Die suksesvolle vestiging van Apis mellifera capensis werkers as sosiale parasiete (pseudo koninginne) in die korwe van die naburige A. m. scutellata, is bewerkstellig deur hul unieke kenmerke, wat met reproduktiewe oorheersing verband hou. Dit suggereer dat die produksie van ʼn tipiese koningin feromoon sein een van verskeie beduidende faktore is in capensis werkers se sukses. In hierdie studie het ons die dilemma van feromoon kommunikasie probeer aanspreek deur te ondersoek of die fenotipiese uitdrukking van seine van die mandibulêre kliere deur genetiese en/of omgewings faktore beïnvloed word. Die hipotese was dat die mandibulêre klier profiele van koninginne en werkers korreleer met spesifieke genotipes in die korf. Die verskillende omgewings waarin werkers groot gemaak word en verouder (sosiale konteks), kan uiteindelik die uitdrukking van gene, raakende mandibulêre kliere, beïnvloed. Dit beklemtoon die feit dat omgewings faktore nie noodwendig onbeduidend is nie. Beide omgewings/sosiale toestande and genotipes van toets individue is in ons eksperimente gemanipuleer. Die capensis werkers afkomstig uit hul natuurlike habitat (Weskaap area) wat in ons eksperimente gebruik is word na verwys as inboorling werkers, terwyl parasitiese capensis werkers, van klonies parasitiese afkoms, vanuit die Gauteng area verkry is. A. m. scutellata werkers was vanuit hul natuurlike habitat, noord van die, hybried sone, verkry. Beide inboorling en parasitise werkers het die potensiaal getoon om reproduktief aktief te word, maar versnelde feromoon ontwikkeling van parasite werkers het hulle ʼn reproduktiewe voordeel gegee. Parasiet werkers het reeds lae hoeveelhede 9HDA, die voorganger van 9ODA, begin produseer tussen 12 – 24 uur, terwyl inboorling werkers produksie eers na 24 uur begin het. Ten spyte van die versnelde ontwikkeling in parasiet werkers het dit nie gelei daartoe dat kloniese parasiete altyd feromonies die oorhand oor inboorling werkers gekry het nie. In groepe bestaande uit inboorling werkers en ʼn enkele parasite werker, was die mandibulêre klier profiele altyd deur 9ODA en 9HDA (> 80% van ekstrakte) gedomineer. Slegs 43% van parasite werkers het groter hoeveelhede 9ODA as inboorling werkers geproduseer. In groepe werkers, wat ʼn groter proporsie gene in gemeen gehad het, het mandibulêre klier profiele konvergeer. Dit ondersteun die bestaan van ʼn verband met genotipiese invloed. Werkers van 75 – 99% verwantskap het beduidend verskil van groepe met laer verwantskapsvlakke, hoofsaaklik as gevolg van die teenwoordigheid van 9ODA (Spearman’s rank korrelasie r = 0.66, p < 0.0001). Ten spyte van die konvergerende neiging van profiele, van meer verwante groepe, was aansienlike veranderlikheid onder verskillende sosiale toestande waargeneem. Werkers, afkomstig vanaf ʼn enkele capensis koninginby, maar òf in die teenwoordigheid òf afwesigheid van ʼn koningin verouder is, het baie kenmerkende mandibulêre klier profiele getoon (Wilks’ lambda λ = 0.118, χ2 = 331.002, p < 0.0001). Die veranderlikheid was dus ʼn gevolg van die sosiale omgewing waaraan die werkers blootgestel was. Dit blyk asof die fisiologiese kenmerke wat met reproduksie potensiaal in heuningbye verband hou, naamlik mandibulêre klier feromoon produksie, deur ʼn kombinasie van genetiese – en omgewings faktore beïnvloed word. Sekere werkers is meer geneig tot die biosintese van koningin mandibulêre klier feromoon as gevolg van hul genetika, terwyl die finale oksidasie na 9ODA onder sterk omgewings invloed is. Die plastisiteit in mandibulêre seine waargeneem in hierdie studie, is aanpasbaar en help werkers om hul reproduksie potensiaal te bereik.

This thesis examines three factors that may influence the change in protein content and size of the brood food glands in honey bees. Effects on the mandibular gland, involved in the production of brood food and in royal jelly, have not been examined in relation to primer pheromones while effects on the hypopharyngeal glands, also involved in the production of brood food, have not been examined in relation to queen mandibular pheromone. This thesis provides preliminary insight into how these pheromones affect the extractable protein content of brood food glands. The first study in this thesis assessed the effects of brood pheromone (BP), queen mandibular pheromone (QMP), and pollen presence on the protein content of hypopharyngeal and mandibular glands of the honey bee. In this study, newly emerged bees were caged for 12 days in one of eight treatments: Queenless state: 1) control (no pollen + no pheromone), 2) pollen, 3) BP, 4) BP + pollen; Queenright state: 1) QMP, 2) QMP + pollen, 3) BP + QMP, 4) BP + QMP + pollen. This study indicated that regardless of pheromone treatment, the most influential factor on gland protein content and size was pollen. The second experiment examined effects of varying pollen dilution on hypopharyngeal and mandibular gland protein content, bee mass, and lipid content of the honey bee. In this experiment, newly emerged bees were caged for 7 days and fed one of five treatments: pollen, 1:1 pollen: cellulose (vol:vol), 1:2 pollen: cellulose (vol:vol); 1:3 pollen: cellulose (vol:vol), and cellulose. This study indicated that bees on the pollen diet were significantly greater than all other diluted diets in measurements of hypopharyngeal gland protein content, lipid content, and mass with significantly less consumption. However, mandibular gland protein content of bees on the pollen diet was significantly greater only from pure cellulose.

A divisão de trabalho é um dos temas centrais no estudo dos insetos sociais. Nas abelhas e nas vespas essa atividade é regulada pelo polietismo etário. Vários autores mostraram que o polietismo etário de Apis mellifera tem ativação seletiva das glândulas com o trabalho feito dentro da colônia, além de diminuição do peso corporal ao começar o forrageio. Nos vespídeos sociais, o polietismo etário é um tema pouco explorado. Com o objetivo de identificar e descrever em Polybia paulista estas mudanças corporais e glandulares relacionadas à idade dos indivíduos ou com o seu uso na função social, analisaram-se operárias de diferentes idades mantidas em cativeiro desde a sua emergência nas quais foi restrita a interação social desde sua emergência e operárias de diferentes idades coletadas desde o ninho onde a interação social foi completa. Operárias de Polybia paulista mantidas em cativeiro aumentaram a massa corporal, diminuíram a massa do mesosoma e diminuíram a secreção das glândulas salivares e mandibular conforme de acordo com o aumento da idade. Isso difere daquelas operárias que foram permitidas do desenvolvimento social normal (grupo controle), onde foi observado um aumento na massa corporal, na massa do mesosoma e na secreção das glândulas salivar e a mandibular em relação ao aumento da idade das vespas. Provavelmente, o relacionamento social estimula a formação e a transição fisiológica relacionada ao polietismo etário em Polybia paulista. Nestas vespas, mudanças corporais e glandulares observadas foram relacionadas com idade, sendo provável que a interação social e o uso das estruturas nas diferentes tarefas dentro da colônia estejam influenciando tais mudanças. Como conseqüência, fatores que atuam sobre a demografia da colônia, como a fase de desenvolvimento ou o bem-estar da colônia, podem ser determinantes na regulação do polietismo etário. Estes resultados contrastam com o modelo proposto para o polietismo etário da abelha Apis mellifera e com observações da atividade glandular na vespa social Polistes versicolor / The division of labor is a central theme in the study of social insects. In bees and wasps this ability is regulated for age castes of the workers. In Apis mellifera, several authors have shown selective activation of the glands for job in colony, and reduced body weight for foraging. In social wasps age polyethism among workers is a relatively unexplored subject. We tried to identify and to describe in Polybia paulista if the bodily and glandular changes are related to the age of the wasps or to the use of the structures in the social function. Workers of different ages at which was restricted social behavior since its emergence and workers of different ages with full social development were analyzed. When Polybia paulista was in captivity, increased body mass, decreased the mass of mesosoma and decreased the salivary and mandibular glands secretion in relation to the age advancement of the wasps. While, allowing normal social development, there was an increase in body mass, the mass of the mesosoma and the salivary and mandibular glands secretion in relation to age. Apparently, social interaction activates the formation and transition of age castes in Polybia paulista. In these wasps the bodily and glandular changes observed were related to age. Probably social interaction and the use of structures within the nest are influencing the direction of such changes. As consequence, factors that influence the demography of the colony, as the development stage or the welfare of the colony, may be decisive in the age castes regulation. These results contrast with the model proposed for the Apis mellifera age polyethism and with observations of glandular activity in the social wasp Polistes versicolor.

A divisão de trabalho é um dos temas centrais no estudo dos insetos sociais. Nas abelhas e nas vespas essa atividade é regulada pelo polietismo etário. Vários autores mostraram que o polietismo etário de Apis mellifera tem ativação seletiva das glândulas com o trabalho feito dentro da colônia, além de diminuição do peso corporal ao começar o forrageio. Nos vespídeos sociais, o polietismo etário é um tema pouco explorado. Com o objetivo de identificar e descrever em Polybia paulista estas mudanças corporais e glandulares relacionadas à idade dos indivíduos ou com o seu uso na função social, analisaram-se operárias de diferentes idades mantidas em cativeiro desde a sua emergência nas quais foi restrita a interação social desde sua emergência e operárias de diferentes idades coletadas desde o ninho onde a interação social foi completa. Operárias de Polybia paulista mantidas em cativeiro aumentaram a massa corporal, diminuíram a massa do mesosoma e diminuíram a secreção das glândulas salivares e mandibular conforme de acordo com o aumento da idade. Isso difere daquelas operárias que foram permitidas do desenvolvimento social normal (grupo controle), onde foi observado um aumento na massa corporal, na massa do mesosoma e na secreção das glândulas salivar e a mandibular em relação ao aumento da idade das vespas. Provavelmente, o relacionamento social estimula a formação e a transição fisiológica relacionada ao polietismo etário em Polybia paulista. Nestas vespas, mudanças corporais e glandulares observadas foram relacionadas com idade, sendo provável que a interação social e o uso das estruturas nas diferentes tarefas dentro da colônia estejam influenciando tais mudanças. Como conseqüência, fatores que atuam sobre a demografia da colônia, como a fase de desenvolvimento ou o bem-estar da colônia, podem ser determinantes na regulação do polietismo etário. Estes resultados contrastam com o modelo proposto para o polietismo etário da abelha Apis mellifera e com observações da atividade glandular na vespa social Polistes versicolor / The division of labor is a central theme in the study of social insects. In bees and wasps this ability is regulated for age castes of the workers. In Apis mellifera, several authors have shown selective activation of the glands for job in colony, and reduced body weight for foraging. In social wasps age polyethism among workers is a relatively unexplored subject. We tried to identify and to describe in Polybia paulista if the bodily and glandular changes are related to the age of the wasps or to the use of the structures in the social function. Workers of different ages at which was restricted social behavior since its emergence and workers of different ages with full social development were analyzed. When Polybia paulista was in captivity, increased body mass, decreased the mass of mesosoma and decreased the salivary and mandibular glands secretion in relation to the age advancement of the wasps. While, allowing normal social development, there was an increase in body mass, the mass of the mesosoma and the salivary and mandibular glands secretion in relation to age. Apparently, social interaction activates the formation and transition of age castes in Polybia paulista. In these wasps the bodily and glandular changes observed were related to age. Probably social interaction and the use of structures within the nest are influencing the direction of such changes. As consequence, factors that influence the demography of the colony, as the development stage or the welfare of the colony, may be decisive in the age castes regulation. These results contrast with the model proposed for the Apis mellifera age polyethism and with observations of glandular activity in the social wasp Polistes versicolor.

La colonie d’abeille (Apis mellifera L.) est une société complexe où les individus interagissent entre eux, notamment par le biais de phéromones. L’étude de cette communication chimique est indispensable à la compréhension des régulations sociales mises en place dans la colonie. Chez l’abeille, plus de 50 substances chimiques avec des effets incitateurs ou modificateurs sur la colonie ont été identifiées. Malgré ces découvertes, de nombreux travaux sont à accomplir pour mieux comprendre ce système de communication particulier.La problématique de cette thèse vise à caractériser l’histoire de vie d’une phéromone majeure l’Oléate d’Ethyle (EO), qui permet d’optimiser l’équilibre nourrices / butineuses dans la colonie. Parallèlement, d’autres recherches ont été entreprises, notamment l’étude de la communication chimique de la reine et du couvain, chez qui seulement deux phéromones ont été identifiées avec des effets pléiotropiques dans la colonie.Nos résultats ont mis en évidence une production variable d’EO par les ouvrières, en fonction de l’environnement de la colonie. La production de cette molécule chimique dans la colonie peut également être modifiée par un stress : des abeilles parasitées par du Nosema spp. ont une production anormalement élevée d’EO. En outre, cette molécule phéromonale est transmise des butineuses vers les nourrices par contact cuticulaire et par le pollen.Pour la compréhension de la communication entre la reine et les ouvrières, nos résultats montrent que la reine utilise d’autres composés phéromonaux puissant en redondance de la QMP pour orienter la construction de cire, le phénomène de cour et l’inhibition des ovaires des ouvrières.Chez le couvain, nous avons identifié un composé phéromonal volatil, le E-ß-ocimène, produit majoritairement par les jeunes larves, inhibant le développement des ovaires des ouvrières et accélérant leur maturation comportementale.Ces études nous ont permis d’avoir une connaissance plus précise de la communication chimique au sein de la colonie. Ainsi nous expliquons par deux théories le rôle de la complexité et de la redondance phéromonale de la colonie d’abeilles / In the honeybee colony (Apis mellifera L.) studies of the chemical communication are essential to understand social regulations. In the honey bee colony more than 50 chemical substances with releaser and primer effects have been identified. Despite years of research on this type of communication, significant work remains to be done.In this thesis, the aim is to characterize the dynamics of a major pheromone: ethyl oleate (EO), which optimizes the balance between nurses and foragers in the colony. In addition, we initiated research on the queen and brood chemical communication in which only two pheromones have been identified in the colony.We have demonstrated that EO production by workers varies under different colony environment. EO production can also be modified by stress; honey bees parasitized by the Nosema spp. have abnormally high EO production. In addition, we identified that EO is transmitted from foragers to nurses by contact (cuticle and pollen).For the queen, our results indicate that the queen uses multiple redundant pheromones (QMP and other unknown compounds), that affect wax construction, retinue behaviour and worker ovary inhibition.For the brood we have identified a volatile pheromone E-ß-ocimene produced mostly by the young larvae to inhibit the development of workers ovaries and accelerate workers’behavioural maturation.With these studies we clarify some aspects of what is known about chemical communication in the honey bee colony. Then we try to explain the role of complexity and redundancy of pheromones in the honey bee colony by two theories