Correlation Analysis Between TeERR And 20-hydroxyecdysone-related Genes In The Gonads Of Yellow-faced Oil Gourd

Teleogryllus emma Ohmachi&Matsuura belongs to the Insecta,Orthoptera,Gryllidae,and Teleogryllus.In recent years,T.emma has been widely farmed because of its use,medicine,edibility,and special economic value.However,the wild crickets overpopulation cause a significant increase in risk on crops,mostly due to climate change and the lack of biological control technology.Therefore,it is very important to understand the regulation mechanism of cricket molting and reproduction,so as to promote the efficiency of special livestock breeding and improve the prevention and control measures in the field.Insect molting and metamorphosis are coordinated by ecdysteroid hormones(EH)and juvenile hormone(JH).The roles of EH in the reproduction of orthoptera insects remain to be further improved compared with JH.EH is further converted into the active form of 20-hydroxyecdysone hormone(20E)after a series of hydroxylation and oxidation processes in target tissues.20E binds to a heterodimer constituted by the ecdysone receptor(EcR)and ultraspiracle protein(USP).20-EcR-USP complex recruits co-activators(i.e.,transcription factor genes Hsc70 and Hsp90),then binds to 20E response elements(EcRE),and triggers a transcriptional cascade,including transcription of the 20E response genes(i.e.,transcription factor genes E74,E75 and HR3),then participates in the regulation of insect molting,metamorphosis and reproduction.Estrogen-related receptor(ERR)as a novel orphan receptor belongs to the nuclear receptor superfamily.ERR is not combined or activated by estrogen,though it is highly homologous with the estrogen receptor(ER).With the assistance of co-activators,ERR directly binds to the ERR response element(ERRE)and estrogen response element(ERE),then modulates the normal ovary and testis differentiation.In addition,the sequence motif 5'-GGTCA-3' is presented in the invertebrate EcRE,which is actually very similar to the ERE motif 5'-AGGTCA-3'.Thus,we speculate that ERR and EcR may be intimately related to each other,and triggers 20E signaling cascade in insect reproduction.In order to analyze the relationship between TeERR and TeEcR and the function of TeERR,TeEcR,TeHsc70 and TeHsp90 in the gonadal development and maintenance of the testicular function.In this study,T.emma was used as experimental insects.After knockdown of TeEcR,TeHsc70 and TeHsp90 expression using RNA interference(RNAi),expression of the TeERR and 20E-related genes were detected by qPCR.All results are as follows:1.The partial cDNAs encoding the T.emma orthologs of RXR,E75B,HR3,Hsc70 and Hsp90 were isolated and characterized in the adult testes,determined patterns of expression in nymphs and adult crickets.Nucleotide sequences alignment showed T.emma RXR,E75B,HR3,Hsc70,and Hsp90 shared high homology to its counterpart of other insects.20E treatments led to suppressed expression of TeEcR,TeRXR,TeE75B,TeHR3,Te1Hsc70 and TeHsp90.Temporal expression analysis showed that TeERR and 20E-related genes covered four stages of gonadal development,from fourth-instar nymphs to adults.The expression of these genes was not always synchronous in the same stage of testicular development,which differed from ovarian development.TeRXR,HR3,TeHsc70,and TeHsp90 were expressed irregularly in gonads of the same developmental stages,while mRNAs encoding TeERR,TeEcR and TeE75B accumulated to higher levels in ovaries than testes.These results demonstrate that expression of the TeERR and 20E-related genes is responsive to the 20E application and involve in regulating the gonadal development and maintaining the ability of the genital gland,especially the ERR,EcR,and E75B mainly regulate the ovarian development.2.Adult male crickets T.emma were divided into the experimental group,negative group,and control group.Crickets of the experimental group were injected with dsTeERR or dsTeEcR,and the negative group received dsEGFP.The efficiency of TeERR and TeEcR-RNAi was detected in the experimental group.Furthermore,the transcription levels,translation levels,morphological characteristics as well as weight were analyzed in the TeERR or TeEcR knocked down testis.Results showed that the expression level of TeERR or TeEcR was significantly down-regulated(n=3,P<0.05)when treated with 2000 ng dsTeERR or dsTeEcR for 48 h.The mRNA and protein expression level of TeERR could be down-regulated(n=3,P<0.05)using TeEcR-RNAi,and vice versa.TeERR and TeEcR were located in spermatogonia,TeERR and TeEcR-RNAi caused morphological changes in testes,but no obvious effect on weight(n=3,P>0.05).These results indicate that ERR and EcR are intimately related to each other.In addition,ERR may be involved in the 20E signal pathway and maintain the function of adult cricket testis.3.Adult male crickets T.emma were divided into the experimental group,negative group,and control group.Crickets of the experimental group were injected with dsTeHsc70,dsHsp90 and dsTeHsc70 + dsHsp90,and the negative group received dsEGFP,then the efficiency of TeHsc70-and TeHsp90-RNAi and expression of TeERR and 20E-related genes were detected in the experimental group after the crickets treated for 48,72 and 96 h.In addition,the content of chapter 3 has found that the mRNA and protein expression level of TeERR could be down-regulated using TeEcR-RNAi,and vice versa.Therefore,the content of this section also examined the expression of TeEcR,TeERR,TeRXR,TeE75B,TeHR3,TeHsc70,and TeHsp90,and explored the relationship between TeERR and TeEcR to the 20E-related genes.Results showed that expression of the TeEcR,TeERR,TeRXR,TeHR3 and TeHsc70 decreased significantly(n=3,P<0.05),as well as increased of the TeE75B and TeHsp90 after TeERR or TeEcR knockdown.Expression of the TeERR,TeEcR,TeRXR,TeE75B and TeHR3 decreased significantly(n=3,P<0.05)after TeHsc70 + TeHsp90 knockdown.Expression of the TeERR,TeEcR,TeRXR,TeE75B and TeHR3 decreased significantly(n=3,P<0.05),and increased of TeHsp90 after TeHsc70 knockdown.Expression of the TeRXR,TeE75B and TeHR3 decreased significantly(n=3,P<0.05),but but no significant influence on TeERR,TeEcR and TeHsc70 after TeHsp90 knockdown.All these results indicate that ERR and EcR are in the upstream of 20E related genes,which are necessary for the testicular development and morphogenesis.Hsc70 can interact with ERR and EcR involving in the regulation of 20E-related genes,E74A,E75B and HR3 expression.4.Exogenous 20E induction and RNAi were used to explore the role of TeERR and 20E response genes in the ovarian development.4th and 6th instar female nymphs were divided into the experimental group,negative group,and control group.In the 20E induction experiment,nymphs of the experimental group were injected with 500 ng 20E,and the negative group received DMSO,then expression of the TeERR and 20E-related genes was detected after treatment 1,3,6,12 and 24 h.In the RNAi experiment,nymphs of the experimental group were injected with dsTeERR or dsTeEcR,and the negative group received dsEGFP,then the efficiency of TeERR and TeEcR-RNAi was detected.Results showed that expression of the TeERR,TeEcR,TeRXR,TeRXR,TeHR3,TelHsc70 and TeHsp90 decreased significantly(n=3,P<0.05),and TeE75B increased significantly(n=3,P<0.05)in 4th and 6th instar nymphs after 20E treatment and TeEcR knockdown.After TeERR-RNAi,expression of the TeERR,TeEcR,TeRXR,TeHR3,TeHsc70 and TeHsp90 decreased significantly(n=3,P<0.05),and TeE75B increased significantly(n=3,P<0.05)in 4th instar nymphs.However,expression of the TeERR,TeEcR,TeRXR,TeE75B,TeHR3,TeHsc70 and TeHsp90 decreased significantly(n=3,P<0.05)in 4th instar nymphs after TeERR knockdown.These results indicate that ERR and EcR were essential for the 20E signal response in ovarian development.5.4th instar male and female nymphs T.emma were divided into the experimental group,negative group,and control group.Crickets of the experimental group were injected with dsTeEcR,and the negative group received dsEGFP,then expression of TeERR and 20E-related genes were detected and compared in male and female nymphs.The results showed that expression of the TeERR,TeRXR,TeHR39 TeHsc70 and TeHsp90 were down-regulated significantly(n=3,P<0.05)in testes and ovaries.Expression of the TeE75B decreased significantly in testes,while it was increased significantly in ovaries.In addition,TeERR,TeEcR,TeRXR,TeE75B and TeHR3 expression was significantly higher in ovaries than testes.TeHsc70 and TeHsp90 expression was significantly higher in testes than ovaries.These results suggest that ERR,EcR,RXR,E75B and HR3 mainly involve in ovarian development,and Hsc70 and Hsp90 mainly involve in testicular development.To sum up,RNAi and exogenous 20E was conducted in T.emma,then expression of the TeERR and 20E-relaed genes was detected in testes and ovaries.Here,relevance of the TeERR and TeEcR was analyzed,and function of the TeERR,TeEcR,TeHsc70,and TeHsp90 in 20E signal cascades was identified.All results suggest that TeERR,TeEcR,TeHsc70,and TeHsp90 involve in 20E testicular and ovarian development,and there are essential genes for the 20E signal pathway in testes and ovaries.