Influence Of Nutritional And Spatial Factors In The Development Of Females Honeybees (Apis Mellifera, Apidae)

The honey bee (Apis mellifera) is a highly eusocial insect. It is known that nutritional factors play a major role in the differentiation between queen and workers. The two play very different roles in the honey bee society and both are indispensable. The complete mechanisms for differentiation between queen and worker remain not clear, however, the most recent discovery is that DNA methylation is implicated in caste determinationIn order to understand the influence of nutritional and spatial factors in the development of female honeybees (Apis mellifera, Apidae) through DNA methylation, we reared larvae in different experimental groups. We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62 at L3,L5,L6; morphological characteristics including body weight at emergence, body length, forewing length, forewing width, proboscis length and the 3rd tergum length of adult bees.The results showed that, when larvae were reared in queen cells, but as the duration when larva reared on royal jelly decreased, Dnmt3 activity, Dnmt3 mRNA expression level, and dynactin p62 methylation level in larval head at L6 were significantly increased. Adults that were reared with a shorter duration of royal jelly showed significantly increased forewing length, forewing width, and proboscis length, however, their body weight, body length, the 3rd tergum length were significantly reduced, compared to those reared with a longer duration of royal jelly. The percentage of queens was significantly lower when larvae were reared with a shorter duration with royal jelly and larvae developed mostly into workers. Workers were then reared with the same diet (worker jelly from 4 day old larvae), but reared in two different sized cells (queen or worker cells). As the cell size decreased. Dnmt3 mRNA expression level, and dynuctin p62 methylation level in larval head at L3,L5 increased significantly. In adults that were reared in worker cells, forewing length, forewing width, and proboscis length all significantly increased. however, their body weight, body length, the 3rd tergum length all reduced significantly, compared to those reared in queen cells. The percentage of workers was significantly higher when larvae were reared with worker cells and larvae developed mostly into workers.Injections Dnmt3 double strandedRNA (dsRNA) into L3 larvae head were well tolerated and led to transient but significant decreases in Dnmt3 activity, Dnmt3 mRNA expression level, and dynactin p62 methylation level.Adult reared from ds-Dnmt3 injected larvae showed significantly increased body weight, body length and the 3rd tergum length.Significantly more larvae developed into queens.From these results, we come to the conclusion that both diet type and cell size contribute to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62. This is the first study to show that cell size also plays a role in influencing larval development when diet is kept the same. Furthermore, we confirm that Dnmt3 plays a key role in honey bee caste differentiation.