| Apis cerana cerana,a exclusive honeybee species in China,has become an essential role of the development of the domestic bee industry,owing to its excellent resistance to pests and diseases,and flexibile adaption to the multi-regional climate and honey-sourceplants,coupled with the low temperature tolerance and the capacity in utilizing sporadic bloom source.The salivary gland is one of the most crucial exocrine gland for honeybee especially worker bees with different divisions of labors,like breeding larva and foraging nectar.Depending on several morphological observation and next genome sequencing techniques,the diffirences appearing in tissue and molecular levels among three developmental stages（new emerging,nursing and forager）were investigated and distinguished in this study,which aim to broaden the field in salivary gland research and identify the mechanism of glandular morphogenesis as well as gene expression responding to the division of labors and developmental periods.The main results were as follows:1.Discripancy in the salivary gland structural characters among three kinds of workers under three developmental stages were observed by means of scanning electron microscope（SEM）and hematoxylin-eosin（HE）stain.The cephalic salivary gland（CSG）stresses the volume and shape of alveoli,diameter of branch duct and distribution of fine trachea.The alveoli of new emerging bees is wizened and flat,which convert into smooth and inverted pear-shaped alveoli of nursing and foragers worker,enlarging the volume and alveolar lumen significantly.Similarly,the glandular cells become more cubic rather than the irregular in the new emerging,with the size of intercuticular space expands.Morphological measurement showed the nursing bee’s CSG branch duct diameter increased by 3.4μm（p<0.05）,which was not significantly different from the foragers.As the working bees’age increased,SEM observation showed there were lots of spherical fine trechea in trachea system attached to the surface of CSG.According to previous researches involving chemical analysis of secretions from CSG and glandular function,many differences formed with the increase of the developmental period reflect that the secretory activity and capacity of CSG gradually increase to meet the functional requirements of the gland under the change of labor division of the worker beesThere were more distinct structural characters that showed defference in thoracic salivary gland（TSG）among three periods.It was new emerging bee that have been observed with less branches of secretory tube（ST）,secretory cell with imcompact cytoplasm,and narrow subcuticular space.But a kind of branch called double secretory tube could be find more frequntely in nursing,with the diameter of ST significantly rises（p<0.05）.Meanwhile,with a large number of vesicles appear in the cytoplasm of secretion cells,the radial subcuticular space extend to the surrounding of secretory cell during nursing bee period.Compared with the nursing bee,foragers almost change little except a few increase involving the diameter of ST（P<0.05）,On the other hand,There were complex treachea cluster structure observing on the connection of outer lobes of TSG and primary tracheas in nursing bee and foragers,which showed more fine trachea adhered with ST than new emerging bee.Therefore,the most discrepancy of TSG between three developmental stages implied the same trendency as the ones in CSG,which support vital structural basis and secretions for different labor activities of workers.2.Based on high-throughput sequencing technique,a RNA-seq programe involving 9 groups worker samples within 3 kinds of developmental stages（New emerging worker,Nursing worker and Foragers）has been acommplished.The total clean date of single group been filtered reach above 66.62GB,while the GC base proportion is around 40%.The percetntages of each groupin which qualification score reach Q30 is betwwen 91.91%and 92.85%.With the whole genome of Chinese bees in the NCBI database（GCA002290385.1）as the reference genome,transcriptome reads acquired from RNA-seq were conduct that map to genome flagment,the mapping rate of reads has been more than 93%,and total 11,324 genes have been identified,including 1,388 novel transcripts according to gene precdiction and annotation.A total of 1,353 differential expression genes were found,of which 20 genes are shared in three comparisons between two groups.The number of DEGs between EM and FG is the highest,with a total of 1085,433 upregulated gene and 652 downregulated genes.This was followed by a total of 752 between EM and NS,309 of the gene expression was raised and 334 were lowered.The number of different expression genes in the first two comparison groups reached 484.Only 98 genes differed between NS and FG,including 35 up-regulated genes,63 down-regulated genes,and around 25 percent of the genes in this comparison group were specific throughout all comparison groups.Analysis of DEGs in transcription groups showed that the salivary glands of worker bees were adapted to changes in the individual development duration and division of labour by changing gene expression:（1）The expression of numerous genes in the oxidation phosphorylation in the salivary glands of worker bees decrease with the increase of age,while the expression of some genes associated with cell proliferation,such as the mTOR signaling pathway,the Wnt signaling pathway and the TGF-beta signaling pathway,which decreased with the increase of the developmental stages as well.It suggest that the salivary gland of worker bees boost energy metabolism level and accelerate cell proliferation and differentiation at a lower age,promoting the development of glandular structure in order to meet the secretion needs of Nursing workers and foragers.（2）The expression patterns of various transformative enzymes genes,as well as the high amount of gene expression of integral membrane protein SV2 and period protein Per,associated with starch and sucrose metabolism,extracellular matrix receptor interaction and circadian rhythm in nursing workers and foragers,which is the genetic basis in determining the salivary gland on participating to forage nectar,as a vital exocrine gland.Furthermore,it assured that workers are able to regulate the capacity and activity of salivary gland,to adapt to the rhythmic changes caused by different division of labor.（3）Depending on the analysis of expression patterns of themembers of the royal jelly protein family and the small peptide Apisimin,it is shown that the salivary gland is likely to be another important Production site of the royal jelly main proteins,with the same trendency of the MRJPs secretion activity of the workers’ hypopharyngeal gland. |
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