| Due to aggregation,migrant and gluttony,Mythimna separata cause a serious threat to crop production.The primary fuel for insect migratory is carbohydrates,lipid and amino acids,In most insects carbohydrates is the major source of energy for short-term fly or take off.During the migration of armyworm,nutrition and environmental temperature are the most important factors.Therefore,in present study,M.separata was used as a model to investigate the effects of supplemental nutrition on sex pheromone biosynthesis.The main results are as following:1.Supplementary nutrition is required for sex pheromone biosynthesis.The contents of trehalose,pyruvic acid and acetyl coenzyme A in the sex pheromone gland(PGs)were significantly increased by feeding with sugar.As a result,sex pheromone biosynthesis was increased,and the ability of females to attract males,mating frequency and fecundity increased significantly.In addition,the re-feeding of starving female moths also resulted in significant increase of trehalose,pyruvate and acyl coenzyme A concentrations in PGs,and finally promoted the production of sex pheromone.In conclusion,supplementation of nutrients contributes to the utilization of trehalose in PGs,which in turn increase the concentration of pyruvate and acyl coenzyme A,the precursor of sex pheromone significantly,and finally lead to increase in sex pheromone production and mating frequency.2.Effect of the starvation on gene expression in PGsA total of 48797129 Unigene sequences were obtained from PGs of females fed with sugar and water.20511,15880 and 13226 sequences were compared in NR database,Swissprot database and KOG database respectively;12667 sequences were annotated by GO protein function,and 6185 sequences were analyzed by KEGG pathway map.There are 5147 unigenes are classied in 5 functional databases,6081 in Go and KEGG databases,only 4649 unigenes were aligned in NR database.Compared with the control fed with water,744 genes were up-regulated and 699 genes were down regulated at 48 h after 5% sugar diet.At 72 h,844 genes were up-regulated and 756 genes were down regulated.3.Screening and functional analysis of Fatty acyl co A reductase(far)Ttranscriptome data of M.separata PGs screen eight FAR.Multiple sequence alignment and evolutionary tree analysis of FAR revealed that the obtanined FAR shared higher identidity with other FARs from other insect species.Real-time quantitative PCR analysis showed that these FAR were expressed different time and in different tissues.FAR2 and FAR9 transcripts showed the higher level expression in ?? tissues,and FAR9 transcripts showed the highest expression level in PGs,indicating that FAR9 play a major role in sex pheromone production.further RNAi-mediated knochdown manifested that the decrease of FAR9 transcript lead to decrease os sex pheromome production,female abilty to attract to males.4.Screening and functional analysis of trehalose transporter(TRE)geneEight gene sequences related to trehalose transporter were obtained from the transcriptome data of M.separate PGs.multiple alignment and evolutionary tree analysis revealed that trehalose transporter in M.separate shared higher sequence identity with other TRE fron other insect species.The transcriptional expression of these eight genes after starvation was detected by real-time fluorescence quantitative technique.The results showed that feeding water with 5% sugar resulted in significant increase of two tre genes(tre1-e and??)in armyworm fed with 5% sugar.Further analysis of developmental and tissue expression patterns showed that the relative expression trend of tre1-e was consistent with the requirement of trehalose in reproductive stage.On this basis,the function of the gene was verified.The results showed that compared with dsegfp injection,the relative expression of tre1-e gene decreased by 36.2%,and the decrease of tre1-e gene expression level led to the significant decrease of trehalose content in PG.These data suggest that tre1-e gene plays an important role in the entry of trehalose into PGs from haemolymph.5.Functional analysis of trehalase geneTrehalase is a key enzyme that catalyzes the hydrolysis of trehalose into two glucose molecules.When the expression level of trehalase gene decreased,trehalose could not be transformed into glucose normally and enter the tricarboxylic acid cycle.After RNAi was used to reduce the expression level of trehalase gene,it was found that the decrease of trehalase gene expression level led to significant increase of trehalose content in PG,finally caused significan decrease of the contents of pyruvate and acetyl coenzyme.These results indicated that although trehalose was transported to PG in large quantities,it could not be effectively converted into glucose,which led to the accumulation of trehalose in PG,in turn cause to decrease in contents of pyruvic acid and acetyl coenzyme,therefore lead to decrease in sex pheromone production,female calling ability and mating frequency.6.The effect of ecdysone on PG immunity.20E titer decreased and then increased after starvation.20 E treatment leads to significant increase of 20 E response genes and antimicrobial peptide genes in vitro.After successful knockdown of Ec R-B,attactin,defensin,cecropin-2 and gloverin were significantly decreased compared with the control.Broad knockdown leads to significant decrease of cecropin-3 transcript.These results indicate that 20 E regulates the expression of PG antimicrobial peptide in response to starvation signal,thus regulating the immunity of PG to the outside world.7.Temperature affects sex pheromone biosynthesisTemperature is the main factor that affects the spatial and temporal distribution of insects,and play important roles in insect growth,development and reproduction.The biosynthesis of sex pheromone in M.separata is regulated by pban.The binding of pban with receptors on the surface of sex pheromone gland cells activates the activity of calcineurin.A series of biochemical reactions take place under the action of can,which ultimately affects the production of z11-16 ald.We detected the contents of trehalose,acetyl Co A and z11-16 ald in the PGs at different temperatures.The results showed that: with the increase of temperature,trehalose and acetyl coenzyme a content increased significantly,ACC,far,and △ 11 desturase gene expression levels all increased significantly with the increase of temperature in the peak period of sex pheromone biosynthesis;the yield of z11-16 ald increased significantly with the increase of temperature at 72 h,but there was no significant difference in the production of z11-16 ald in the appropriate temperature range at 95 h,which were significantly higher than that at 31 ℃ There was no significant difference between 19 ℃ and 25 ℃ in total fecundity,and both were significantly higher than 31 ℃.However,the peak of oviposition began to advance with the increase of temperature.The speed of ovarian development also increased with the increase of temperature.From these data,we draw the following conclusion: appropriate high temperature is conducive to the development of ovary;at the same time,the content of acetyl coenzyme A,the precursor of sex pheromone synthesis,can be increased by increasing trehalose in PG,and the enzyme activity of ACC enzyme and the transcription level of genes related to sex pheromone synthesis will be promoted,and the rate of sexual information biosynthesis and mating success will be promoted. |
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