Evaluation of differential gene expression during behavioral development in the honeybee using microarrays and northern blots

BACKGROUND: The honeybee (Apis mellifera) has been used with great success in a variety of behavioral studies. The lack of genomic tools in this species has, however, hampered efforts to provide genome-based explanations for behavioral data. We have combined the power of DNA arrays and the availability of distinct behavioral stages in honeybees to explore the dynamics of gene expression during adult development in this insect. In addition, we used caffeine treatment, a procedure that accelerates learning abilities in honeybees, to examine changes in gene expression underlying drug-induced behavioral modifications. RESULTS: Spotted microarrays containing several thousand cDNAs were interrogated with RNAs extracted from newly emerged worker bees, experienced foragers and caffeine-treated bees. Thirty-six differentially expressed cDNAs were verified by northern blot hybridization and characterized in silico by sequencing and database searches. Experienced foragers overexpressed royal jelly proteins, a putative imaginal disc growth factor, a transcriptional regulator (Stck) and several enzymes, including α-glucosidases, aminopeptidases and glucose dehydrogenase. Naive workers showed increased expression of members of the SPARC and lectin families, heat-shock cognate proteins and several proteins related to RNA translation and mitochondrial function. A number of novel genes overexpressed in both naive and experienced bees, and genes induced by caffeine, have also been identified. CONCLUSIONS: We have shown the usefulness of this transcriptome-based approach for gene discovery, in particular in the context of the efficacy of drug treatment, in a model organism in which routine genetic techniques cannot be applied easily

MicroRNAs in Honey Bee Caste Determination

The cellular mechanisms employed by some organisms to produce contrasting morphological and reproductive phenotypes from the same genome remains one of the key unresolved issues in biology. Honeybees (Apis mellifera) use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Here we investigate the honeybee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. We show significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers). Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signalling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Our data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required

Light-inducible transcriptomic and epigenomic changes underlying brain plasticity in honeybees

Dissertação de Mestrado apresentada à Faculdade de Letras da Universidade de Coimbra.Pretendemos, com esta dissertação, fundamentar a categorização como ritual laico da Arte como Veículo, propósito da última fase do trabalho de Jerzy Grotowski, discutindo os modos particulares dessa categorização. No primeiro capítulo expomos, resumidamente, o percurso artístico de Grotowski e as principais influências que marcaram o seu pensamento. Explicamos os conceitos fundamentais que estruturam a sua prática artística e ensaiamos uma definição para a Arte como Veículo. Finalizamos o capítulo com uma discussão sobre a categorização como ritual das criações performativas produzidas segundo este modelo. No segundo capítulo abordamos o ritual como performance e discutimos a origem ritual dos géneros estéticos performativos. Apresentamos definições operativas de ritual e performance e abordamos os principais aspectos do ritual enquanto performance, recorrendo sobretudo à Antropologia da Performance de Victor Turner. No final do capítulo exploramos a categorização da Arte como Veiculo como ritual, à luz da Antropologia da Performance. No terceiro capítulo fazemos um estudo sumário de um ritual tradicional, o culto dos Theyyams do Norte Malabar, na Índia. Contextualizamos e descrevemos o ritual e algumas das suas particularidades. Relatamos as impressões recolhidas durante um estudo de campo na região, à luz da experiência da Arte como Veículo e dos aspectos realçados pela Antropologia da Performance. Concluímos com a enumeração de resultados obtidos e a confrontação entre o que se apurou na investigação sobre a Arte como Veículo e as impressões recolhidas no estudo do Theyyam, apontando-se desenvolvimentos que possam futuramente confirmar ou reforçar os resultados agora apresentados.With this dissertation I intend to justify the categorization as a secular ritual of the Art as Vehicle, the last phase of Jerzy Grotowski's work, discussing the particular ways of this categorization. In the first chapter I briefly expose the artistic course of Grotowski and the main influences that marked his thought. I explain the fundamental concepts that structure his artistic practice and I essay a definition for Art as Vehicle. I end the chapter with a discussion of the categorization as a ritual of performing works created according to this model. In the second chapter I address the ritual as performance and briefly discuss the ritual origin of performative aesthetic genres. I propose operative definitions for ritual and performance and I enumerate the main aspects of ritual as performance, calling upon, mostly, the Anthropology of Performance from Victor Turner. At the end of the chapter I explore the categorization as ritual of Art as Vehicle at the light of the Anthropology of Performance. In the third chapter I make a summary study of a traditional ritual, the worship of Theyyams in the North Malabar, India. I contextualize and describe the ritual and some of its special features. I report the impressions gathered during a field study in the region, at the light of the experience of Art as Vehicle and of the aspects highlighted by the Anthropology of Performance. I conclude with an enumeration of results and the confrontation between what was found in the research on Art as Vehicle and the impressions gathered in the study of Theyyam, pointing out future developments that may confirm or strengthen the results now presented

Epigenetic regulation of the honey bee transcriptome: unravelling the nature of methylated genes

Background: Epigenetic modification of DNA via methylation is one of the key inventions in eukaryotic evolution. It provides a source for the switching of gene activities, the maintenance of stable phenotypes and the integration of environmental and genomic signals. Although this process\ud is widespread among eukaryotes, both the patterns of methylation and their relevant biological roles not only vary noticeably in different lineages, but often are poorly understood. In addition, the evolutionary origins of DNA methylation in multicellular organisms remain enigmatic. Here we used a new 'epigenetic' model, the social honey bee Apis mellifera, to gain insights into the significance of methylated genes.\ud \ud Results: We combined microarray profiling of several tissues with genome-scale bioinformatics and bisulfite sequencing of selected genes to study the honey bee methylome. We find that around 35% of the annotated honey bee genes are expected to be methylated at the CpG dinucleotides by a highly conserved DNA methylation system. We show that one unifying feature of the methylated genes in this species is their broad pattern of expression and the associated 'housekeeping' roles. In contrast, genes involved in more stringently regulated spatial or temporal functions are predicted to be un-methylated.\ud \ud Conclusion: Our data suggest that honey bees use CpG methylation of intragenic regions as an epigenetic mechanism to control the levels of activity of the genes that are broadly expressed and might be needed for conserved core biological processes in virtually every type of cell. We discuss the implications of our findings for genome-scale regulatory network structures and the evolution\ud of the role(s) of DNA methylation in eukaryotes. Our findings are particularly important in the context of the emerging evidence that environmental factors can influence the epigenetic settings of some genes and lead to serious metabolic and behavioural disorders

Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera

<p>Abstract</p> <p>Background</p> <p>In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown.</p> <p>Results</p> <p>By using cDNA microarray analyses of >6,000 <it>Apis mellifera </it>ESTs, we found 240 differentially expressed genes (DEGs) between developing queens and workers. Many genes recorded as up-regulated in prospective workers appear to be unique to <it>A. mellifera</it>, suggesting that the workers' developmental pathway involves the participation of novel genes. Workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater proportion of physiometabolic genes, including genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., <it>tor</it>). Many DEGs are likely to be involved in processes favoring the development of caste-biased structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents. Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of caste development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented <it>cis</it>-elements from four gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between <it>cis</it>-elements and genes and objectively quantify the degree of relationship between these entities.</p> <p>Conclusion</p> <p>We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees. This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. Finally, we propose a conceptual model of caste differentiation in <it>A. mellifera </it>based on gene-regulatory networks.</p

Contrasting Sex-and Caste-Dependent piRNA Profiles in the Transposon Depleted Haplodiploid Honeybee Apis mellifera

Protecting genome integrity against transposable elements is achieved by intricate molecular mechanisms involving PIWI proteins, their associated small RNAs (piRNAs), and epigenetic modifiers such as DNA methylation. Eusocial bees, in particular the Western honeybee, Apis mellifera, have one of the lowest contents of transposable elements in the animal kingdom, and, unlike other animals with a functional DNA methylation system, appear not to methylate their transposons. This raises the question of whether the PIWI machinery has been retained in this species. Using comparative genomics, mass spectrometry, and expressional profiling, we present seminal evidence that the piRNA system is conserved in honeybees. We show that honey bee piRNAs contain a 2'-O-methyl modification at the 3' end, and have a bias towards a 5' terminal U, which are signature features of their biogenesis. Both piRNA repertoire and expression levels are greater in reproductive individuals than in sterile workers. Haploid males, where the detrimental effects of transposons are dominant, have the greatest piRNA levels, but surprisingly, the highest expression of transposons. These results show that even in a transposon-depleted species, the piRNA system is required to guard the vulnerable haploid genome and reproductive castes against transposon-associated genomic instability. This also suggests that dosage plays an important role in the regulation of transposons and piRNAs expression in haplo-diploid systems.This study was supported by the Australian Research Council DECRA grant DE13010