The Molecular Mechanisms Of Nutrition And Larvae Inducing On Hypopharyngeal Gland Development Of Apis Mellifera

In apicultural process, adequate supply of pollen is an important guaranty for workers to product RJ. Meanwhile we often find that the larvae can strongly stimulate nurse bee to secret RJ. In other words, the RJ production is strongly relied on the nutrition and existence of larvae. But the molecular regulation mechanism of the development of HGs is still unclear. In this study, we investigated the molecular regulation mechanism of the effects of nutrition and existence of larvae on the HGs development using histocytological analysis, molecular biology and bioinformatics methods. The corresponding results will help to elucidate the molecular mechanism of RJ secretion of worker bees. The main research contents and results are as follows: 1. The effects of nutrition and larvae inducing on the HGs development of Apis mellifera honey beesTo explore the effects of pollen and larvae inducing on the HGs development and RJ secretion of A. mellifera honey bees, 9 healthy colonies, from sister-queen with similar population were chosen, and randomly divided into 3 groups(named Hon, Pol and Bro, respectively), each group contained 3 colonies. Hon, Pol and Bro were feed with abundant honey, honey and rape pollen, and honey, rape pollen and larvae, respectively. New emergence workers were marked for sample collecting, and the experiment was conducted 12 days. The HGs of 6-day old, 9-day old and 12-day old workers were separated, the content of protein was tested by BCA Protein Quantitation Kit; the ultrastructure of hypopharyngeal gland cells of 9-day old worker were detected by transmission electron microscope; IlluminaHiSeqTM 2500 paired end sequencing technology was used to test the transcriptome expression of HGs of 9-day old workers. The results are as follows:(1) Pollen could significantly improve the protein content in HGs for 6-day old and 9-day old workers. Both pollen and larvae could stimulate the protein synthesis in HGs of 9-day old workers, and the Bro group was statistically significant better than the Pol group(P<0.01).(2) The size order of cell endocrine tubes was Bro group, Pol group and Hon group; the rough endoplasmic reticulum of Bro group and Pol group were presented as long tubular or small bubble shape, which containing a large number of ribosomes in the surface, while the rough endoplasmic reticulum of Hon group contained less ribosomes.(3) The bioinformatics analysis of the transcriptome sequencing results showed that a total number of 7 types of variable shear and 87,792 SNP were detected, and a total of 360 new genes were found, of which 230 genes could annotate to the COG、GO、KEGG databases. Furthermore, 2,801 differentially expressed genes(DEGs) were detected, the differential genes between Hon group and Pol group, Hon group and Bro group, Pol group and Bro group were 220, 2,380 and 1,658,,respectively. And including 213, 2,328 and 1,617 the number of annotationed genes were 213, 2,328 and 1,617, respectively. Pathway enrichment analysis showed that the genes associated with larvae inducing were mainly involved in the biological process of oxidative phosphorylation and Wnt signaling pathways. The nutritional specific genes were mainly involved in the peroxidase, fructose and mannose metabolic activity pathways. Further, the genes associate with nutritional and larvae inducing signals were mainly involved in the ribosomal protein, oxidative phosphorylation, TOR signaling pathways and branch chain amino acid metabolism. We paid special attention to 9 mrjps genes, the cluster analysis results of these genes showed that mrjp4 and mrjp7, mrjp5 and mrjp8 were two close function genes clasters. 2. Functional analysis and validation of candidate genesThirty-six DEGs among Hon group, Pol group and Bro group were gained using venn analysis to excavate the important genes that regulating the development of HG. Among these genes, mrjp2, mrjp3, Loc724114 and Loc410337 were selected as the typical genes for the further discussion. They were all induced due to the up-regulated expression in a high level by pollen and larvae factorstreatments, and the expression levels were the largest. Therefore, we chose these 4 genes as candidate genes to study their expressing characteristics.Total of 1,980 honey bee workers were collected and randomly divided into 6 groups(named A, B, C, D, E and F, respectively) with 3 duplication, and each duplication contained 110 honey bees. All bees were reared in wooden boxes at the incubator for 15 days. Groups A and B were feed with honey, honey and rape pollen, respectively. Group C was applied with a piece of comb contained larvae based on Group B. Group D was feed with honey for the first 9 days, and added rape pollen for the next 6 days., Group E was applied with a comb containing larvae based on Group D. Group F was feed with honey and rape pollen for the first 9 days, and applied with a comb containing larvae for the next 6 days. First, at the 1st, 3rd, 6th, 9th and 12 th day, 10 bees was collected in groups A, B and C. Total RNA was isolated from the head, thorax muscle, cuticle, midgut and poison gland, and then they were validated by qRT-PCR to determine the levels of the candidate genes. Next, at the 9th day, 30 bees of A, B and C groups were selected; and at the 15 th day, 30 bees of group A to group F were collected to determine the expression of candidate genes in HGs. The results indicated that:(1) The gene Loc724114 and Loc410337 were induced expression by larvae+pollen in the head and midgut of 9-day-old worker bees, meanwhile the gene mrjp2 and mrjp3 expressed mainly in head, muscle and cucitle of 9-day-old worker bees.(2) The pollen and larvae+pollen could significantly induce the expressionof Loc724114, Loc410337, mrjp2 and mrjp3 at 9th days, but the expression was significantly reduced at 15 th days(P<0.01).(3) Gene Loc410337 and Loc724114 were regulated mainly pollen+larvae; and mrjp2 and mrjp3 were superimposed regulated by pollen and larvae.This study indicated that nutrition and brood inducing stimulated organelles increased of HG cell, improving the ability of tissue protein synthesis, and promoted the development of worker bee HGs. Nutrition and brood inducing effected transcription of HG genome. The DEGs mainly by Ribosome pathway positive regulation and TOR, Wnt signaling pathway negative regulatory role in the combined effect on worker bees HG development. Pollen(nutrition) is essential nutrient during HG development of worker bees, and brood presence is the key incentives to promote the development of HG, so nutrition and larvae are two key factors in the regulation of inducible HG development.