Reproductive Sharing In Honeybee Colonies

Apis mellifera, a eusocial insect with advanced division of labor, is not only an economic insect with long keeping history, but also an emerging model organism. Eusocial insects are characterized by reproductive division of labor among female individuals. Normally, honeybee colony is monogynous and reproduction in honeybee colony is monopolized by the queen. Reproductive conflict among queens is settled via swarming, destroying queen cells or lethal fighting. Workers, with few exceptions, do not reproduce in queenright colonies with existence of young brood. In the cases that workers reproduce in queenright colonies, eggs laid by workers are policed by other workers, which is termed worker policing. As a result, only about 0.1% of males are the sons of workers in normal western honeybee colonies.However, there are two kinds of colonies being of special reproductive division of labor. They are artificially created multiple queen colony, in which several queens coexist and share reproduction, and the colony of Apis mellifera capensis, in which it is usual that workers reproduce and workers have the potential to act as social parasites. During parasitizing, they enter the colony of other subspecies, share reproduction with the host queen and finally usurp the reproduction of the host colony.Taking the advantages of the reproductive features of multiple queen colony and A. m. capensis honeybee colony, the reproductive sharing in honeybee colonies was studied. The contents and major results are listed as follows.(1) The effect of mandible ablation on queens'fighting strategy in the initial stages of creating multiple queen colonies was studied by behavioral observing. The results showed that mandible ablation of queens had significant effect on their decision on fighting strategy and weak queens refrain from engaging in lethal contests based on self-assessment, but not mutual assessment of fighting ability.(2) Data collected from 18 apiaries in which multiple queen colonies were used revealed that the technique to create multiple queen colonies based on biological induction and environmental induction was applicable in beekeeping practice with high ratio of success (78.1%). The majority of such multiple queen colonies were kept for 6 months without queen loss and 55.8% of them overwintered successfully without queen loss. These results indicated that such multiple queen colonies were sustainable enough for long-term keeping and, in other side, overwintering period was sensitive to them. Moreover, mandible ablation did not have significant effect on queen's egg laying capacity. Egg laying capacity of multiple queen colonies (3-queen colony and 5-queen colony) was significantly higher than that of single queen colony. The results laid a solid foundation for the application of multiple queen colonies in beekeeping practice.(3) Reproductive output and mandibular gland pheromone of individual queens in multiple queen colonies were studied. Data showed that reproductive output of each queen in multiple queen colonies was unequal and that the average output of coexisting queens in the colonies was significantly lower than that of queens in single queen colonies and was negatively correlated with the amount of queens coexisting in the colonies. Analysis on mandibular gland pheromone revealed that queens of multiple queen colonies had significantly higher amount of 9-oxodec-2-enoic acid (90DA) than that of queens of single queen colonies, which hinted pheromonal competition occurred among coexisting queens. No significant difference on mandiblular gland pheromone and on the amount of retinue workers was found among queens of multiple queen colonies, which indicated the disproportional reproductive output of them was not due to the possible pheromonal inhibition, at least as far as mandibular gland pheromone was concerned.(4) Gene expression profiles of the heads of queens of multiple queen colonies and single queen colonies were compared by microarray analysis. Statistical analysis with MultiExperiment Viewer V4.3 found 45 down-regulated genes and 7 up-regulated genes, of which AM05325and AM07925 were related with important metabolic pathways. AM05325 was involved in biosynthetic process of unsaturated fatty acids, the precursor of mandibular gland pheromone components; AM07925 was involved in biosynthesis of terpenoid and steroids. Up-regulated expression of gene involved in biosynthesis of unsaturated fatty acids further confirmed the pheromonal competition among queens in multiple queen colonies.(5) Taking the advantage of multiple queen colonies, the role of mandibular gland pheromone in regulating worker reproduction was tested by analyzing the correlation between the absolute and relative amount of mandibular gland pheromone components and reproductive output of queens, and between the amount of retinue workers and reproductive output of queens. No high correlation was found. This result indicated that mandibular gland pheromone was not an indicator of reproductivity of queens, but more possibly acts as the direct inhibitor of the development of workers ovaries.(6) Mandibular gland profiles of queenright A. m. capensis workers sampled from four locations were analyzed and compared with that of A. m scutellata workers. The data showed that mandibular gland pheromone of A. m. capensis workers in core distribution area of this subspecies was dominated by (E)-9-hydroxy-2-decenoic acid (9HDA), a precursor of queen substance 90DA, with small amount of 10-hydroxy-2-decenoic acid (10HDA) and 10-hydroxydecanoic acid (10HDAA). A. m. capensis workers near the introgression zone of A. m. capensis and A. m scutellata had less 9HDA and 90DA than workers in core distribution area, but more than A. m scutellata workers. This result indicated that reproductive control system in A. m. capensis colonies is unique when compared with other honeybee subspecies, which could explain why workers of this subspecies engaging in egg-laying faster than other subspecies in the absence of a queen and could represent a pre-adaptation to social parasitism.To sum up, I studied the mechanism based on which reproductive sharing was achieved in multiple queen colonies, the practicability of application of multiple queen colonies in beekeeping practice, relationship among queens in multiple queen colonies, differential gene expression caused by coexisting of queens and the role of queen mandibular gland pheromone in regulating worker reproduction. Furthermore, mandibular gland pheromone of queenright A. m. capensis workers was studied.The studies of multiple queen colonies on egg-laying ability and sustainability are of significance in understanding the biological features of multiple queen colonies and promoting their application in beekeeping practice. Moreover, studies on reproductive sharing based on multiple queen colonies revealed the mechanism to achieve reproductive sharing among queens and some characters related with reproductive sharing in honeybees. The study on queenright A. m. capensis workers revealed the pheromonal character related with reproductive sharing between queens and workers, which is of significance in learning the regulation and evolution of social context in insects.