The Molecular Mechanisms Of Nutritional-cross Breeding In Apis Cerana Body Color Regulated By Apis Mellifera’s Royal Jelly

Honeybees Apis mellifera and Apis cerana are two major honeybee species that are extensively maintained and examined.Previous studies have shown that these species could alter their morphology,physiology,and,behaviors by exchanging their larval diets,namely honeybee nutritional crossbreed.However,its underlying epigenetic mechanisms remain unclear.This thesis aimed to explore the effects of Apis mellifera’s royal jelly on the development of Apis cerana queens and its underlying epigenetic mechanisms.This study has implemented an in vitro rearing technology to rear Apis cerana-Apis mellifera nutritional crossbreeding queens.The results showed that only a low rate of Apis cerana larvae could develop into nutritional crossbreed queens(NQ,2.08～33.30%),which has been influenced by seasons.Similar results were found in Apis mellifera queens(only 4.78-27.90% of larvae could develop into queens.Feeding a 50 μL larval diet twice a day had the highest survival rate compared to the 25 μL,100 μL and 150 μL groups(P <0.05).Via adding a plastic queen cell into 24* cell culture resulted in a significantly higher newborn weight of NQs compared to the 24* cell culture group,as well as a larger thorax(P <0.05).Rearing full nutritional crossbreed Apis cerana queens using the above method,and rearing Apis cerana queens in their native colonies as a control group(CQ).The body colors of NQs and CQs were measured under a microscope,and their gene expression was compared using RNA-Seq.The results showed that the body color of NQs turned yellow compared to CQs,with a significantly lighter color in its scutum,scutellum,tergum,and sternum.Totally,1484 differentially expressed genes(DEGs)were identified between NQs and CQs,of which seven key DEGs were enriched in three melanin synthesis pathways and dopamine.Knowing down of DEGs(TPH1 and KMO)resulted in a change in body color,confirming their functions in honeybee body color formation.The biased expression of non-coding RNAs between NQs and CQs were compared using whole RNA sequencing technology.The results showed that 311,92,and 169 significantly differentially expressed DElnc RNA,DEmi RNA,and DEcirc RNA,respectively.Interestingly,eight DElnc RNAs and three DEmi RNAs were identified as participating in the gene regulation of the above seven key DEGs.These results indicated that non-coding RNA participating in Apis cerana-Apis mellifera nutritional crossbreed by regulating the expression of critical genes,resulting in dramatic changes in honeybee body color.By using DNA methylation sequencing,the different DNA methylation levels between NQs and CQs were compared.The results showed that 993 significantly differentially DNA methylated regions were identified,in which three key DMG(OGDH,ALDH,and ALDH7)were enriched into three key pathways of melanin synthesis.The ALDH and ALDH7 were the same as the seven key DEGs participating in melanin synthesis.Consequently,these findings revealed that DNA methylation also participates in Apis cerana-Apis mellifera nutritional crossbreed as an important epigenetic modification.In conclusion,this study demonstrated that Apis cerana-Apis mellifera nutritional crossbreeding results in dramatic body color alternation in Apis cerana queens.Non-coding RNAs and DNA methylation act as vital epigenetic modifications in this process by regulating the expression of key genes,resulting in color changes in the honeybees.The findings of this thesis showed a clear genetic and epigenetic mechanism of nutritional-crossbreed between Apis cerana and Apis mellifera,which not only enriches our understandings on honeybee biology and molecular biology but also serves as model for studies in phenotypic plasticity and its epigenetic mechanisms of other animals and plants.