Proteomic Analysis Of Honeybee (Apis.mellifera) Caste Determination

A honeybee (Apis mellifera L.) colony is a highly organized insect society consisting of two castes; asingle queen, thousands of workers. In spite of same genetic makeup, the queen and the workers showalternative morphologies, behavior and physiology. The female queen is large in size and specializes inreproduction, while workers are small and engage in colony maintaining activities. Their life spans also varythat the queen lives for1-2years, while the workers only live6-7weeks. Existing information indicate thatthis alternative morphology, behavior and physiology are driven by nutritional difference during their3,5days old larval stage. Despite the successive investigations on the underlying causes of this honeybee castepolymorphism, information at proteome levels that considers early developmental stages (less than3.5daysold larvae) is limited. In this study, we analyzed the caste determination mechanisms of the queen and theworker destined larvae using mitochondrial and nuclear proteomes at72,96and120hours and through theirtotal proteome at48hours developmental stages. Combinations of differential centrifugation, twodimensional electrophoresis, mass spectrometry, bioinformatics and quantitative real time PCR were applied.There were significant qualitative and quantitative protein expression differences between the two castes atthree developmental stages both at mitochondria and nuclear levels. Interestingly, the queen-destined larvaeupregulated large proportions of proteins at all the developmental stages from both sub-cells. In particular,the queen larvae upregulated95%of the mitochondrial and69%of nuclear located proteins at72hours.Although wide-ranging mitochondrial proteomes participate to shape the larvae metabolic, physiologic andanatomic differences between the two castes at72hours, physiometabolic-enriched proteins (metabolism ofcarbohydrate and energy, amino acid and fatty acid, protein biosynthesis) as well as protein folding were foundas the major modulators of the profoundly marking of this caste differentiation. As well, the prospectivequeen larvae exclusively up regulated most of the nuclear enriched proteins (cytoskeleton, development andnucleic acids) that have nuclear functions to regulate DNA and RNA activities during the process of casteformations. The proteins differential expressions from both subcellular enriched were further verified byfunctional enrichment and biological interaction network analyses as a direct link with metabolic rates andcellular responses to hormones and DNA/RNA functions. In general, the changing mitochondrial and nuclearproteome of the two castes intended larvae indicate that the two larvae are on different trajectories as early asbefore72hours and further recommended a research works that considers the larvae age less than72hoursold. Further research attempt of comparing the two caste intended larvae with differential protein expressionat48hours indicate the queen intended larvae upregulated60%of the total47identified proteins. Thissuggests that the two larvae have already on different trajectories at48hours. To our knowledge, these firstsubcellular and this early stage global proteomic data explore the innermost biological makings of honeybeesociety’s polymorphism and pave way to other eusocial insect caste pathway decision mechanisms. Inparticular, the global proteome results significantly advance the time of caste decision to48hours. This is amajor step forward in the analysis of the fundamental causes and mechanisms of honeybee caste pathwaydecision and greatly contributes to the knowledge of honeybee biology. In particular, the consistency between the proteins and mRNA expressions from the subcellular proteomes provides us important targetgenes for the reverse genetic analysis of caste pathway modulation through RNA interference.