Screening Of Key Genes Associated With The Mandibular Gland Secretion Biosynthesis Of Western Honey Bee (Apis Mellifera) And Their Regulation By Age And Nutrition Factors

Mandibular gland is an important honey bee exocrine gland,its secretions are of great importance in maintaining the social organization of honey bee colony.Stearic acid is used as the synthetic precursor in biosynthesis pathways of both queen and worker mandibular gland secretion,but the caste differences in the following fatty acid?-oxidation process lead to the different proportions of components of mandibular gland secretions between castes.Queen mandibular gland secretion mainly consists of 9-oxo-2-decenoic acid(9-ODA)and possesses multiple functions including attracting workers and drones,inhibiting activation of worker ovaries,etc.Worker mandibular gland secretion is dominated by 10-hydroxy-2-decenoic acid(10-HDA)and 10-hydroxydecanoic acid(10-HDAA),constituting an important part of royal jelly.Identifying candidate genes associated with mandibular gland secretion biosynthesis will provide insights into the molecular mechanism of mandibular gland biosynthesis and the molecular basis of the caste difference in mandibular gland.In this study,by using high-throughput RNA-sequencing technology,we compared the gene expression patterns of mandibular glands from mature queens,normal workers obtained from queenright colonies and ovary activated workers obtained from queenless colonies.Aiming to contributes to the description of the molecular basis for the biosynthesis of fatty acid-derived pheromones in the mandibular glands.Then we identified the specific function of certain P450 in mandibular gland using expression pattern and RNA interference technology.10-HDA is a major secretion of honey bee workers' mandibular gland and the key component of royal jelly.It is the most important marker in royal jelly that determines the price of royal jelly in commercial trade.Thus,it is of great value to elucidate the secretion pattern of honey bee workers' mandibular glands and its regulation.In this study,the expression level of genes associated with 10-HDA biosynthesis in the mandibular glands from workers in different ages were evaluated.In addition,we analyzed the gene expression pattern of workers' mandibular gland feeding different diet,in order to understand the effect of age and nutrition on honey bee workers'mandibular gland.The main results were summarized below:(1)mRNA associated with mandibular gland biosynthesis.Secretions from mandibular glands have important caste-specific functions that are associated with the social evolution of honey bees.To gain insights into the molecular architecture underlying these caste differences,we compared the gene expression patterns of mandibular glands from queens,queenright workers and queenless workers using high-throughput RNA-sequencing technology.In total,5457 differentially expressed genes were identified among these groups.Of these,2629 were differentially expressed between queens and queenright workers,2761 between queenright and queenless workers,and 4662 between laying workers and queens.Multiple differentially expressed genes were found to associate with the caste specific mandibular gland secretion biosynthesis,participating in the hydroxylation of fatty acid,chain-shortening of fatty acid and the modification of terminal hydroxy group,which provide important clues for clarifying the mechanisms underlying the mandibular gland production of fatty acids derived components.Our results unravel the molecular basis of mandibular gland biosynthesis and provide new insights into the molecular mechanisms involved in mandibular gland functions.(2)identification of cytochrome P450 related to mandibular gland biosynthesis.In the mandibular gland biosynthesis pathway,cytochrome P450 is responsible for the hydroxylation of stearic acid.In order to identify the specific P450 involved in this process,we analyzed the expression levels of six P450 genes that were most abundantly expressed in the transcriptome analysis across different castes,ages,tasks and tissues.Our analysis revealed that CYP6AS8 and CYP6AS11 are selectively expressed in the mandibular glands of workers and queens compared to other tissues.In addition,we knockdown the expression level of CYP6AS8 in the mandibular glands of workers by using RNA interference.We found that the 10-HDA content in the mandibular gland is closely associated with the expression level of CYP6AS8.Our result suggested that CYP6AS8 and CYP6AS11 might be responsible for the critical bifurcated hydroxylation process in the biosynthesis pathway,providing important clues to description of the molecular basis for the caste difference in mandibular gland secretion biosynthesis.(3)the expression pattern of genes associated with 10-HDA biosynthesis in the mandibular glands from workers in different ages.As the major 10-HDA secretion organ,verifying the worker mandibular glands'secretion ability is a significant contribution to the beekeeping industry.In order to elucidate the relationship between the secretion ability of mandibular gland and the age of worker,we evaluated the expression level of genes associate with 10-HDA biosynthesis in the mandibular glands from workers of different ages,including CYP6AS8,CPTI,ACOX1,ACOX3,CSP3 and mrjpl.We found that except for mrjpl,the expression levels of all these gene were not affected by worker's age,indicating that worker mandibular gland's secretion ability will not fall as age growth.(4)The effect of diet on the biosynthesis ability and gene expressions of honey bee workers' mandibular glands.Honey bee's diet has a significant impact in the secretion ability of exocrine glands.By regulating the nutrition level of caged workers' diet,we measured the effects of diet on the secretion ability and the gene expression pattern of workers' mandibular gland.Gas chromatography analysis revealed that workers fed on insufficient nutrition produce significant less 10-HDA,while the expression levels of genes associate with 10-HDA biosynthesis were upregulated in RNA-sequencing analysis.Taken together,this study explores the molecular basis of mandibular gland biosynthesis and verifies the cytochrome P450 involved in the synthesis of 10-HDA.These will contribute to further studies about the molecular mechanisms of caste difference in mandibular gland function.The effects of age and nutrition on the gene expression of mandibular gland from worker are evaluated,providing clues to the regulation of the secretion ability of worker's mandibular gland.