The Role Of Insulin Signaling Pathway In The Regulation Of Termite Lifetime Based On Transcriptome Sequencing

Although different castes of eusocial insects like bees,ants and termites live in the same colonywith different lifespan and some castes may differ by two orders of magnitude.Termite reproductive castes?queens and kings?can live for 20 years,while non-reproductive castes of workers and soldiers live for weeks to months.Besides,all individuals in the termite population have the same genetic background,and the difference in life span determined by the difference in gene expression,which leads to the abnormal life and sustained reproductive capacity of reproductive-caste queens and kings.Insulin is a protein hormone that controls the metabolism of sugars,fats and proteins through signal transduction in cells,affecting growth and development processes such as reproduction and aging.Among organism such as nematodes,fruit flies,yeast,rodents and other higher animals,glucose signaling mechanisms are highly conserved.The prolonged life span was previously reported in Caenorhabditis elegans and Drosophila melanogaster by defective insulin signal transduction.However,there is no report about the insulin signaling pathway and life span regulation in termites.In the present study,Reticulitermes chinensis was used to determine the insulin signaling pathway and established artificial colonies from swarmer alates.The inaugural colony became a complete experimental test colonies after four years.Therefore,we used Illumina Hi Seq^TM4000 platform to perform transcriptome sequencing analysis of the primary king?PK?,primary queen?PQ?,male worker?WM?,and female worker?WF?of the R.chinensis.Moreover,we compared related genes to insulin signaling pathway between different castes.The real-time quantitative PCR method was used to verify the accuracy of the transcriptome sequencing results.By analyzing the differences in insulin signaling pathway-related gene expression and regulation of termites life span among different castes of R.chinensis.The main research conclusions are as follows:?1?The Reticulitermes chinensis termite colonies were reared for four years.There was a significant difference in the abdominal width and body length of the primary queen and primary king to their female and male workers.However,there were no significant differences in the abdominal width and body length of workers of the same sex at the same age.The primary queen has obvious physogastry.?2?The Illumina Hi Seq TM 4000 platform was used to sequence the transcriptome of the R.chinensis primary termite queen and king,male and female workers.During the transcriptome analysis a total of 161,933 unigenes were obtained,with a total base number of 109,126,456,the shortest unigene length was 201 bp,while the longest unigene length was 19,482 bp,the average length of all unigenes was 673 bp,GC content was 42.90%and N50 is 933.?3?The transcriptomic sequence of the R.chinensis among the primary king and primary queen,male worker and female worker consists of 161,933 unigenes,of which the Nr database is annotated to 60,736 unigenes;the KOG database is annotated to 27,181 unigenes,involving a total of 25 functional classifications;the Swissprot database annotates to 31,699unigenes;the KEGG database annotates to 19,291 unigenes,involving 343 biological signaling pathways;the annotated unigenes of the GO database involved in biological processes,cellular components and molecular functions.A total of 59 physiological and metabolic functions are related to the annotation of the Nr function database.The R.chinensis transcriptome sequence data is nearly related to the Zootermopsis nevadensis.?4?A total of 9,357 simple repeats?SSRs?were identified from the transcriptome data of the primary queen of the R.chinensis,the primary king,male and female workers and distributed in 8,194 sequences of the dinucleotide.The number of acids is 3,403,trinucleotides are 4,245in numbers,tetranucleotides are 1,124 in numbers,pentanucleotides are 178 in numbers and the number of hexanucleotides was 407.The identification of these SSRs can help research on genomic differences and genetic map construction among species.?5?According to the different significance criteria?the differential gene expression change is more than 2 times,and the p-value?0.05?,the number of different genes between the primary king and the primary queen are 1,026,733 genes were up-regulated and 293 genes were down-regulated.The number of different genes between the primary king and the male worker was 26,170,with 24,503 genes up-regulated and 1,667 genes down-regulated.The number of different genes between the primary queen and the female worker was 69,511,with55,671 genes up-regulated and 13,840 genes down-regulated.The number of different genes between male and female workers was 1,926,with 1,160 genes up-regulated and 766 genes down-regulated.The GO enrichment analysis of differential genes of the transcriptome sequence of the primary queen of the R.chinensis,the primary king,male and female workers distributed in the cellular process and catalytic activity,metabolic process,cell part,single-organism process and binding.?6?There are two central pathways in the insulin signaling pathway,namely the extracellular signal-regulated kinase?ERK?/mitogen-activated kinase?MAPK?pathway and the PI3K-Akt pathway.Among them,the PI3K-Akt pathway is the main insulin signaling pathway of insect.Therefore,we selected six related genes in the PI3K-Akt signaling pathway for real-time quantitative PCR to verify the results of transcriptomic sequencing data.The real-time quantitative PCR results revealed that the differential gene expression was consistent with the transcriptome sequencing results.The correlation coefficient between was 0.721,indicating that our transcriptome sequencing data is authentic and reliable used for subsequent biological analysis.?7?The expression levels genes showed that Pdk1 expression was low in the primary queen and king,but there were no significant differences between different castes?n=3,P=0.04<0.05?.The results of q RT-PCR showed that the expression of akt2-a of the primary queen and king was lower than that of the female worker?n=3,P=0.025<0.05?.Also,the expression of Tsc2 in the primary king was higher than that of the non-reproductive castes?n=3,P=0.05?0.05?.In contrast,m TOR expression in the primary king and the primary queen was lower than that of male and female workers?n=3,P=0.019<0.05?.In addition,the expression levels of EIF4E?n=3,P=0.015<0.05?and RPS6?n=3,P=0.016<0.05?were significantly lower in the primary queen and king than in male and female workers.It is speculated that the low expression of the four genes akt2-a,m TOR,EIF4E,and RPS6 may inhibit the malignant proliferation of cells by inhibiting the invasion and metastasis of cancer cells,so that the primary queen and king have a longer life.Tsc2 is a tumor suppressor gene.High expression in the reproductive castes will also inhibit the occurrence of malignant cell proliferation in the reproductive castes.Besides,there is no significant difference between different castes of Pdk1,which also indicates that there may be differences in insulin signal regulation between the R.chinensis and other model organisms.