Genetic Differentiation And Ceramide Metabolic Enzymes Related To The Development Rate Of Chilo Suppressalis

Chito suppressalis(Walker),commonly known as rice stem borer,is one of the most important rice pests that distributes in various rice fields in China.In recent years,the occurrence of C.suppressalis has increased with increasing insecticide resistance,overwintering climate and farming system.The development state of the organism affects the survival and reproduction of population.Pests can improve the internal stability of the population structure by regulating the development process of the individuals and populations.The study of internal stability mechanisms will provide a new theoretical basis for the development of pest control systems.This study was based on genetic differentiation and ceramide metabolic enzymes to explore the mechanisms that affecting the development rate of C.suppressalis.We constructed the genomic DNA libraries of the fast and slow growth rate of C.suppressalis by using Double digest restriction-site associated DNA sequencing(ddRAD-seq)technology and then sequencing,the genes affecting the development rate were investigated and analyzed for the study of the genetic diversity of the C.suppressalis.The RNAi technology was used to interfere the key genes of alkaline ceramidase(saCER)and neutral sphingomyelinase(snSMase)which ralated to the developmental rate of C.suppressalis,and their roles in sphingolipid metabolism and development rate of C.suppressalis were primarily explored.The main results are as follows:1.The ddRADseq method was used for the flrst time to construct genomic DNA libraries of C.suppressalis,which has extremely fast development and extremely slow phenotypic differentiation.Through sequencing and bioinformatics analysis,a total of 61513 SNPs were obtained,and the Bayescan statistical analysis included a total of 130 outliers.Analyzing the functions of candidate genes corresponding to outliers,we speculated that aminopeptidase,glycogen synthase,metabotropic glutamate receptor,diuretic hormone receptor,glutathione S-transferase,and NADH ubiquinone oxidoreductase may be related to the development rate of C.suppressalis.2.The RNAi technical system for silencing the saCER and snSMase genes,the key genes for the development rate of C.suppressalis,was established by micro-injection and nanoparticle-mediated feeding.The interference efficiency of the two methods were compared and the results showed that the saCER and snSMase genes are sensitive to RNAi by intoducing dsRNA.After optimizing the conditions,the RNAi efficiency of saCER gene had the highest level(41%)at 60 hours post-injection with the dsRNA concentration of 5000 ng/μL,the snSMase gene had the highest RNAi efficiency at 48 hours post-injection(47%)with the dsRNA concentration of 2500 ng/μL,and the saCER and snSMase genes had the highest RNAi efficiency on the 7th day(32%)and the 8th day(52%)respectively after feeding with(48 μg/d).There was no significant difference in RNAi efficiency between saCER gene and snSMase gene using the same introduction method.The same gene of C.suppressalis,saCER or snSMase,showed no significant difference in the interference efficiency between methods of micro-injection and the nanoparticle-mediated feeding.The protocols of RNAi via injection and feeding which developed and optimized in this study is feasible in the basic research of enzyme genes of the lipid metabolism and can be used for the subsequent study of the sphingolipid metabolism of the C.suppressalis.3.On the basis of interfering the C suppressalis larvae saCER and snSMase genes,the ceramides content were measured by Quadrupole Liquid Chromatography Tandem Mass Spectrometry(LC-MS-MS)after interfering the saCER and snSMase genes,in order to clarify the physiological roles and regulation mechanisms of saCER and snSMase.The results showed that inhibition of the expression of the saCER gene increased the level of C.suppressalis ceramides,and the C18sCer increased significantly.The level of C.suppressalis ceramides decreased while inhibiting the expression of the snSMase gene.The results showed that the saCER and snSMase genes can effectively regulate the level of C.suppressalis ceranides,and thus affect the sphingolipid metabolism and regulate the development rate of C.suppressalis.In summary,this study has investigated genes that may be related to the developmental rate of C.suppressalis in the genetic level,but the specific genes function verification and regulation mechanisms need to be further explored.On the level of sphingolipid metabolism,we clarified that the alkaline ceramidase gene(saCER)and the neutral sphingomyelinase gene(snSMase)can affect the sphingolipid metabolism by regulating the level of Csuppressalis ceramides and thus affecting their development rate.