RNA-seq-based Differential Analysis Of Intestinal Gene Expression In Chinese Honey Bee At Different Developmental Stages

Compared with Apis mellifera,Apis cerana cerana has many advantages such as strong individual cold resistance,strong mite resistance,spawning quantity control,anti-tea honey poison,etc.and its various bee products also have extremely high economic and nutritional value.The honeybee goes through four stages of egg,larvae,pupa and adult.During the metamorphosis of workers,through literature review and anatomical observations,there are obvious differences in the external morphology and internal structure of the digestive tract,howerver the cause and mechanism of the differences are not clear.This study selected 7 developmental periods of honeybee works:6-day-old larvae(WL),prepupa(WP),white-eye pupa(WW),pink-eye pupa(WF),red-eye pupa(WR),black-eye pupae(WB)and adult(WA),set three biological repeats for each period.Based on RNA-seq sequencing to study the gene expression of the digestive tract transcriptome level,to explore the molecular mechanism of workers intestinal development.The main results are as follows:1.Complete transcriptome sequencing of 21 digestive tract samples to obtain 136.51 Gb Clean Data,each sample reaches 5.93 Gb,Q30 is not less than 93.22%,and GC content is about 40%.The clean reads were compared with Apis cerana cerana reference genome,and the comparison efficiency of 19 samples reached more than 90%.2.After repetitive correlation assessment,principal component analysis and cluster analysis,WP3 samples in the prepupa stage and WA2 samples in the adult stage are eliminated.With FC≥ 2 and FDR<0.01 as the standard,the samples of the previous stage in each adjacent group were used as the control group to screen differentially expressed genes,and finally the DEGs of WL-WP;WP-WW;WW-WF;WF-WR;WR-WB;WB-WA respectively.were 2535(up 1309,down 1044),307(up 69,down 238),140(up 43,down 97),570(up 450,down 120),537(up 379,down 158),3200(up 1394,down 1806).The Venn diagram found that there were a total of 980 DEGs in the two metamorphosis periods of WL-WP and WB-WA,and different changes under the control of the same molecular mechanism occurred.3.Analyze the DEGs between adjacent groups in sequence.GO enrichment results show that the entries annotated into the three branches of each group are basically similar,all of which are enriched into cellular and metabolic processes,biological regulation,cell membranes and their components,cells and their components,binding,catalytic activity,etc.Combined with the KEGG annotation results,several pathways related to cell proliferation,metabolism,energy metabolism,photoreceptor system,immune function were discovered:①Cell proliferation and tissue development:Annotated gene expression changes and cluster analysis of the extracellular matrix receptor interaction pathway,Wnt signaling,Hippo signaling,and MAPK signaling pathway were shown,the changes of Laminin,Agrin,Wnt,F-actin,PKC and other proteins related to cell,tissue,muscle,and nerve development are consistent.The pupal stage is a critical period for the proliferation and differentiation of the digestive tract cells and the formation of tissues(the prepupal stage was the most active).In addition,the level of reactive oxygen species controlled by Duox protein increased,the wing morphology controlled by Omb protein begins to occur,but the Rac protein was inhibited at this stage,indicating that the cell transfer and polar transport were not carried out during the prepupa,it was more inclined to increase the number of cells based on the original structure.The dMekkl protein associated with ultraviolet radiation,bacterial infection,heat shock,and hypertonic pressure in the digestive tract of adult worms is activated,and the level of intestinal immune function and stress resistance is improved compared with the larval and pupal stages.②Metabolism and energy metabolism:By analyzing the gene expression changes annotated in the pathways of carbon metabolism,oxidative phosphorylation,starch and sucrose metabolism,pentose,glucuronic acid conversion,amino acid biosynthesis,etc,it was found that the changes of NADH dehydrogenase,cytochrome C oxidase and other proteins were similar,the metabolism and energy supply of the digestive tract presents a tendency to decline significantly,then recover,and finally increase with the development of worker bees(prepupa is the lowest point of metabolism),including synthesis and conversion of various amino acids,sugars,fats,etc.,various transmembrane transport,ATP hydrolysis,starch sucrose decomposition supply,etc.This is related to the fact that the bees completely stopped eating during the pupa stage,but the adult bees performed a lot of work on honeycomb cleaning and pollen collection.③Photoreceptor system and eye development:During the prepupa period,the TRPL protein on light transmission is activated,and the coding genes such as Actin and myosin NINAC are up-regulated,and the photoreceptor system and muscle tissue of the eye are initially formed;During the.period of white eye pupa,another TRP protein in the photosensitive channel was activated,and the photosensitive system was well developed.The originally active PKC,Actin,NINAC,and TRP were inhibited in adult bees,and eye development basically stopped.However,the activation of rhodopsin was not found in the whole process,and the exact development time point of the visual system could not be determined.④Tissue lysis and immune function:the activation of related proteins did not appear in the lysosome,especially in the prepupa stage,its production and hydrolysis were inhibited,However,GO enrichment results and morphological data show that there are still many cell decomposition and tissue lysis phenomena,presumably due to a certain protein,enzyme or special physical and chemical changes.Secondly,the production and function of phagosomes were also inhibited,and they began to form at the end of the pupa stage,proving that there was no phagocytosis in the digestive tract of the pupa stage.Combined with dMekkl protein in the MAPK signaling pathway,it can be seen that the adult’s digestive tract immune function is enhanced,and the detoxification mechanism is reduced.At the same time,the hemoglobin level and the stratum corneum structure are well developed,and the intestinal tract is more tough and elastic.The weakened immunity of larvae and pupae is more susceptible to diseases,and beekeepers should pay more attention to the hive environment and body health at this stage in actual production.⑤Circadian rhythm:Per and Cyc proteins in the circadian rhythm during the black-eye pupae are activated,which may prepare for the coming out of the room.In adult bees,the up-regulated genes of this pathway are further increased,the Dbt protein is activated,but the Tim protein has not changed during this process,and the related genes in the digestive tract may not be comprehensive.