|dc.description.abstract||Honeybees (Apis mellifera) are remarkable creatures with a complex eusocial hive structure. There are two female honeybee castes within a hive; a single queen and thousands of workers. The only reproductively active female is the queen. However, should the hive lose its queen (removing the pheromone she produces, queen mandibular pheromone, which keeps the workers in a reproductively inactive state), the ovaries of worker honeybees differentiate from the quiescent state they are normally in and begin to produce eggs.
This remarkable remodelling of adult tissue in response to a changing environment occurs rapidly (within ten days) and is accompanied by changes in the expression of thousands of genes. It was hypothesised that this rapid response may be facilitated by the genome being organised into functional domains, known as gene complexes, containing a number of neighbouring genes which were all upregulated or repressed together to produce the rapid morphological and functional changes observed within the worker honeybee ovary.
This study began by investigating whether one of the three known honeybee gene complexes was involved in worker ovary activation. The Enhancer of Split Complex [E(Spl)-C] is expressed in response to Notch signalling and as Notch signalling plays a role in Drosophila oogenesis this complex was chosen for investigation by RT-qPCR.
This study found that the canonical E(Spl)-C genes were regulated in the same manner as each other during worker ovary activation, although the Tubulin-Tyrosine Kinase-like gene which is inserted within the canonical E(Spl)-C and which is co-ordinately regulated with these genes in development), is expressed in an opposite manner to the rest of this genomic region during worker ovary activation. The identification of a CCCTC-binding factor (CTCF) binding site within this region implies that changes in the three dimensional structure of the DNA may be responsible for the Tubulin-Tyrosine Kinase evading the co-ordinate gene regulation in this region.
To test whether there were other, previously uncharacterised, gene complexes involved in worker ovary activation an RNA-Seq data set which compared the expression of all known honeybee genes between queen ovary, quiescent worker ovary and active worker ovary was analysed using the existing programme REEF (REgionally Enriched Features of genomes). When REEF was found to be limited in its approach, a new programme was written (using the C++ programming language) to identify novel gene complexes expressed within the honeybee ovary. Overall, there was less evidence of association ￼between gene activation and genomic co-localisation than would be expected by chance, however there were some regions identified as having coordinate regulation.
Both REEF and the programme written for this project identified a region on chromosome 2 which had seven neighbouring genes all identified as homologues of Drosophila Lethal (2) – Essential For Life [l(2)efl] which were co-ordinately regulated with respect to ovary activation. This Lethal Complex and the neighbouring genes were investigated using RT-qPCR and the experimental findings confirmed that all genes in this region are regulated in the same way with highest expression being in the ovaries of worker bees. Phylogenetic and bioinformatic analysis identified that this grouping of l(2)efl genes is also present in a number of other hymenoptheran species. Nothing is known about the molecular functions of l(2)efl, yet their duplication and presence in a range of species suggests they are functionally important. This is a region of the genome worthy of further, functional investigation.
One additional region of the honeybee genome was chosen for analysis as it held a group of genes which had highest expression in queen ovary (according to the RNA-Seq data set) and contained a number of genes involved in cell cycle regulation. Many of the Drosophila homologues of these genes are involved in oogenesis or female meiosis. The Cell Cycle group was also analysed using RT-qPCR, the findings of which were consistent with the RNA-Seq data set.
Overall, the honeybee genome is randomly arranged with respect to ovary activation. This makes regions with conserved, coordinated gene expression particularly interesting due to their rarity. Further investigation is required to determine how these coordinated regions are regulated.||