Function Of Micro Rnas In The Regulation Of Reproductive Diapause In Galeruca Daurica

Diapause is a state in which insect growth,development,and reproduction stagnate,and it plays an important role in the life history of insects.With the progress of molecular biology and genomics research technology,the research on the mechanism of diapause regulation has risen from the determination of conventional physiological indicators to the level of molecular mechanism,especially the diapause related genes and their metabolism and signaling pathways have become the focus of research.Current researches suggest that changes in gene expression drive the diapause progress in insects.However,many details of the regulation networks that initiate,maintain,and terminate diapause remain unclear,and almost all is unknown about how non-coding RNAs,including micro RNAs（mi RNAs）,regulate diapause.Galeruca daurica is a new insect pest in the grasslands of Inner Mongolia in recent years,which not only causes great losses to animal husbandry in Inner Mongolia grasslands,but also poses a serious threat to ecological security in north China.This pest is an obligatory diapause insect,which oversummers in an adult diapause form and overwinters in an egg diapause form.This study for the first time conducted the high-throughput sequencing on mi RNAs of G.daurica adults at different diapause stages,screened out a large number of differentially expressed mi RNAs,and studied the functions of two main differentially expressed mi RNAs,which not only helps more deeply reveal the molecular mechanism on summer diapause of G.daurica adults,but also it plays a necessary foundation for further research on the roles of mi RNAs in the regulation of insect diapause.The main results are as follows:1.Using the high-throughput sequencing technology,small RNA sequencing was performed on samples of G.daurica adults at different diapause stages（pre-diapause,PD;diapause,D;post-diapause,TD）,and 222 mi RNAs were identified,including 135 conserved mi RNAs and 87 novel mi RNAs.A time-series cluster analysis showed that 88 mi RNAs were down-regulated during diapause and up-regulated before and after diapause.2.By analyzing the small RNA sequencing database of G.daurica adults at different diapause stages,35 differentially expressed mi RNAs were screened,among which 30 and13 mi RNAs were differentially expressed in the D/PD and TD/D comparisons,respectively.The KEGG and GO analysis of genes targeted by differentially expressed mi RNAs showed that mi RNAs may regulate reproductive diapause in G.daurica mainly by regulating juvenile hormone,ribosome,MAPK signaling,and Ca²⁺signaling pathways.3.By combining bioinformatics analysis,expression profiling,and dual luciferase reporter assays,it was confirmed that the juvenile hormone primary response gene Kr-h1was a target gene of let-7-5p.The injection of the let-7-5p agomir in pre-diapause female adults inhibited the expression of Kr-h1,delayed ovarian development,increased lipid accumulation,promoted fat body development,and induced reproductive diapause.Depleting Kr-h1 by RNAi triggered the same effects.In contrast,the injection of the let-7-5p antagomir led to the opposite results.In addition,the JH agonist Methoprene down-regulated let-7-5p expression and rescued the ovarian developmental defects caused by let-7-5p overexpression.The above results indicated that the JH–let-7-5p–Kr-h1 regulatory network plays important roles in the regulation of reproductive diapause in G.daurica.4.Using a combination of bioinformatics analysis,expression profiling and dual luciferase reporter assays,it was confirmed that the forkhead transcription factor Fox O is a target gene of mi R-2765-3p.The overexpression of mi R-2765-3p in pre-diapause female adults decreased the transcription of Fox O,reduced lipid accumulation,inhibited fat body development,promoted ovarian development,and delayed the entry of diapause.Knocking-down of Fox O by RNAi also achieved the same results.In contrast,the inhibition of mi R-2765-3p resulted in the opposite results.Hormone injection experiments showed that the JH agonist Methoprene promoted the expression of mi R-2765-3p,but ecdysone 20E had no effect.Furthermore,juvenile hormone rescued ovarian developmental defects caused by inhibiting mi R-2765-3p.The above results indicated that JH–mi R-2765-3p–Fox O regulatory network also played vital roles in regulating reproductive diapause in G.daurica.