Study On The Role Of Juvenile Hormone Pathway Related Genes In Termite Soldier Differentiation

Juvenile hormone is a terpenoidcompound that mainly functions in maintaining the physiological status of larvae and regulating insect development,metamorphosis and reproduction.In termites,the addition of exogenous JH can induce the termites to produce excessive soldier ants,resulting in the imbalance of the population grade and the extinction of the population.Therefore,it can be usedas a pesticide.Its specific induction effect andchanges in JH pathway-relatedgenes are not yet clear.Reticulitermes aculabialis as the research object,to JHⅢ and JHA induction differentiation soldier relatedgene expression changes were analyzed,and the change of JH synthetic gene HMG Coa-R after intervention of relatedgene expression changes were analyzed,and to explore the termites soldier JH channel function in the process of differentiation.The specific results are as follows:1 JH Ⅲ and JHA soldier induced resultsFirstly,the mortality andinduction of termites were calculatedby setting the gradient change of culture density and water content,and the conditions of artificial culture were optimized.The results showed that the mortality of termites was the lowest and the induction rate was the highest when the culture density was 60 per plate and the amount of water added was 600 μL,indicating that this condition was the best induction condition.In different seasons with 80 μg JHⅢ induced termites,results show that the mortality and the induction from high to low:March≈October>July.>D ecember.Termites treated with 120 g JHA in different seasons haddifferent effects.The mortality:June>April>October.The induction:June≈April>October.Anyhow,JHⅢ in death andinducedability for workers is higher than in spring and summer,JHA in death andinducedability for workers is higher than in spring and summer.Different concentrations of JHⅢ and JHA processing termites,mortality and the induction from high to low arrangement are as follows:200 μg>160 μg>120μg>80 μg>40 μg.Mortalitys between the two analysis results show that the JHA is better than the stability of JHⅢ.Analysis for induction,according to the results of termite resistant to JHA’s thresholdfor 120-160 μg,JHⅢ within the adding quantity is 200 μg,inducing rate increased with the increase of adding amount of JHⅢ.2.Identification of JH signaling geneWith JHⅢ induction before the workers,a pre-soldier,the soldier three groups of samples induced the transcriptome sequencing.The Unigene 74190 were spliced,and 20,440 Unigene were matched to the NR database.A total of 20985 Unigene were annotatedby NR,KOG,GO and KEGG databases.The statistical results of differentially expressedgenes showed that worker were up-regulatedby 2,916 genes anddown-regulatedby 3,322 genes,which compared with pre-soldier;worker were up-regulatedby 4828 anddown-regulatedby 2065,which compared with the soldier ants;pre-soldier were up-regulatedby 6,874 genes anddown-regulatedby 4,390 genes.Among which the soldier and the pre-soldier had the most differentially expressedgenes(11,264).According to the annotations of Unigene and the current literature reports,86 Unigene related to JH were selected.Are involvedin JH synthetic gene has HMG Coa R(Hyd roxymethylglutharyl-Coa R),MD D(D iphosphomevalonate decarboxylase),Ld FPPS(farnesyl pyrophosphate synthetase),JHE(Juvenile hormone esterase);JH downstream genes are Hexamerin,Met(Methoprene tolerant),Tai(Taiman/p160/SRC/ncoa-like molecule),Kr-hl(Kruppel homolog 1),Broad,Clock(Clock),Cycle and Cry2),PIF(Patency Inducing Factor),Hairy,Groucho,JHBP(Juvenile hormone binding protein),PLC(phospholipase C),CaMKⅡ(calcium/calmodulin-dependent protein kinase Ⅱ),PKC(protein kinase)C)Hsp83(Heat shock protein 83),USP(ultraspiracle),SSP(soldier special protein).3.JH gene expression pattern analysisTwo kind s of reagents(JHⅢ and JHA) respectively with termites,sampling at different time points,with Realtime PCR to detect gene expression patterns.The same concentration of JHⅢ induced termites and JHA,Met,Kr-h1,Br,USP the SSP and HMG-Coa R expression patterns are basically the same.Met reaches its peak on the 5th day after molting.JHA under the action of the expression of Kr-h1 trend relative to JHⅢ expression trendin ad vance;The expression trend of Br was opposite to that of Kr-h1,and the expression trend of Kr-h1 was delayedunder the action of JHA.In JHⅢ and JHA induced,Met,Kr-h1 and Br are of a similar expression patterns in the pre-soldier has a peak around 5 dafter molting,Met,Kr-h1 and Br in JH signal pathway was closely.Under the condition of JHⅢpre-soldier after molting of 0-1 dand JHA under the condition of 0-1 d,7-9 dexpression of significant differences,but differences in multiples.In the pre-soldier 1-7 dafter molting,under the condition of applying JHⅢ exogenous and JHA,SSP gene expression patterns of the same,but to 9d,in JHⅢ conditions,SSP expression quantity up-regulated;in JHA condition,the SSP expression quantity down-regulated;The expression pattern of HMG-Coa R in JHⅢ and JHA under the condition of the same.Three concentration gradients of JHA(120 g,160 g,and 200 g)were used to treat the pre-soldier,and then the expression patterns of Met,kr-h1,Br,USP,SSP,and HMG-Coa R were detectedby Realtime PCR.The results showed that high concentration of JHA could promote the ad vance of SSP gene expression pattern.At the same concentration of JHA,the patterns of Met,Kr-h1 and Br genes were very similar.The expression levels of Met,kr-h1,Br,USP,and HMG-Coa R genes all increasedfrom 7d to 9dafter the first molting of pre-soldieer treated with 200 gJHA,suggesting that they may be to differentiate into soldier,so the gene expression levels all increased.When using JHA to induce differentiation of soldier,the expression patterns of HMG-Coa R and SSP in spring and winter were basically the same,but the variation multiples were different,suggesting that these two genes were closely related to differentiation of soldier.4 HMG-Coa R gene interferenceAfter the interference of HMG-Coa Rgene by dsRNA feeding,it was found that the expressions of Met,kr-h1,Br,USP and SSP genes all decreased.Since HMG-Coa R is a key enzyme for JH synthesis,the interference of HMG-Coa R lead s to the inhibition of end ogenous JH synthesis,so the expression of downstream genes decreased.