Comparative Analysis Of Transcriptome In Different Development Stages Of Dastarcus Helophoroides

Dastarcus helophoroides is an important natural enemy of many forest pests,especially long-horned beetles.The biological control of longicorn beetle by D.helophoroides is safe,efficient and sustainable.Under the suitable feeding conditions,D.helophoroides can live for several years,and can continue sexual reproduction.Its particularity provides special resources for studying the physiological and molecular mechanism of insect reproduction.In order to explore the molecular mechanism of the growth and development of D.helophoroides,we used Illumina novaseq^tm6000 to sequence the transcriptome of D.helophoroides at six different developmental stages:the4th instar larva（L1）,the 6th instar larva（L2）,the pupa（L3）,the one-year adult（L4）,the two-year adult（L5）and the four-year adult（L6）.The abundant transcriptome information of D.helophoroides was revealed.The main conclusions are as follows:（1）18 samples of D.helophoroides（6 different developmental stages,3 repeats each）were sequenced.The number of raw reads obtained from each sample ranged from16155301 to 20624868,and the number of clean reads obtained after quality control ranged from 15599491 to 20179992.The average proportion of filtered clean reads to raw reads was 97.79%.HISAT software was used to compare the clean reads of each sample with the reference genomes of D.helophoroides,and the comparison results were 77.88%-90.38%,more than 70%,which proved the reliability of the test results.（2）In this study,the FPKM values of all genes in 18 libraries of D.helophoroides were statistically analyzed.The data of 18 transcriptome libraries of D.helophoroides were compared by using Deseq software（a total of 15 comparison groups）,so as to screen out differentially expressed genes.The screening criteria was FDR<0.05.The screening results of differentially expressed genes in each comparison group were:4380（L1 vs L3）,1825（L2 vs L1）,2916（L2 vs L3）,2909（L4 vs L1）,2516（L4 vs L2）,2547（L4 vs L3）,5565（L5 vs L1）,5187（L5 vs L2）,5621（L5 vs L3）,1620（L5 vs L4）,5015（L6 vs L1）,4839（L6 vs L2）,5277（L6 vs L3）,1221（L6 vs L4）and 30（L6 vs L5）.Among them,the number of differentially expressed genes was the least between 2-year-old and 4-year-old adults,and the most between pupal and 2-year-old adults.The more the number of differentially expressed genes,the greater the difference of gene expression patterns（3）Pairwise comparison between samples will produce 15 comparison groups,and GO function annotation will be performed for 10 representative groups.Annotation results are divided into three categories:biological process,cellular component and molecular function.The number of significantly enriched entries in each group was 118（L1 vs L3）,35（L2 vs L3）,95（L4 vs L3）,136（L5 vs L3）,137（L6 vs L3）,66（L4 vs L1）,122（L5 vs L1）,98（L6 vs L1）,45（L6 vs L4）,0（L6 vs L5）.For each group,there are differences in terms of biological processes,cellular components and molecular functions,as well as the number of terms.In general:biological process>molecular function>cell component.（4）The number of KEGG metabolic pathways enriched in each group was 109（L1 vs L3）,107（L2 vs L3）,103（L4 vs L3）,107（L5 vs L3）,106（L6 vs L3）,110（L4 vs L1）,112（L5 vs L1）,111（L6 vs L1）,79（L6 vs L4）and 7（L6 vs L5）.In this study,we found several metabolic and signal transduction pathways that play an important role in the growth and development of D.helophoroides in different groups:amino acids,fatty acids,nucleotides,riboflavin and TGF-βSignal pathway,etc.Each pathway is involved in the special growth and development process of D.helophoroides at different developmental stages,which provides a basis for the subsequent analysis of the whole life cycle of D.helophoroides from the molecular direction.（5）Twenty nine differentially expressed genes,including juvenile hormone（JH）,ecdysone（Ecd）,growth factor（GF）,G protein（GP）and nuclear receptor（NR）,were screened from the transcriptome database of D.helophoroides.RT-qPCR results showed that the change trend of qPCR results of most candidate genes was consistent with that of RNA-seq results,which verified the reliability of transcriptome.Most genes such as JH-5,Ecd-4,Ecd-5,GF-1,GF-2,GP-2 and NR-2 were differentially expressed,and some of them were specifically expressed in pupal stage.In this study,we sequenced the transcriptome of D.helophoroides at different developmental stages,screened out the differentially expressed genes in each comparison group,and analyzed their GO and KEGG enrichment,to explore the key genes and various pathways related to the growth and development of D.helophoroides.RT qPCR technology was used to study the real expression trend of candidate genes,which provided a theoretical basis for revealing the growth and development mechanism of D.helophoroides.