Response Of Juvenile Hormone And Ecdysone Of Male Sitotroga Cerealella(Olivier) To Diallyl Trisulfide

Sitotroga grainella Olivie is one of the most destructive pests of stored grains.In the previous studies,we found that the treatment of males with diallyl trisulfide（DAT）(LC₁₀=0.01μL/L)could inhibit the oviposition of female without affecting its mating behavior.Further studies showed that the spermatophore in a mated female transferred from DAT-treated males is significantly smaller than that from controls,and the sperm count in the spermatophore is significantly reduced,and the sperm motility is also significantly inhibited.The results showed the reason that DAT inhibited the oviposition of males might be related with the reproduction of male,but the specific mechanism of DAT was not clear till now.In view of the important regulatory role of the insect endocrine system on insect reproduction,in this thesis we took male S.cerealella as the research object,fumigates the males with DAT(LC₁₀=0.01μL/L)for 7 hours,then respectively determines the protein content of reproductive organs（accessory gland,ejaculatory ducts,testis）and the sperm count（testis,seminal vesicles,ejaculatory ducts）,juvenile hormone and ecdysone titer and the expression of related genes,and finally verifies the functions of the two hormones by exogenous administration of hormone analogs.The main results are as follows:1.Response of male juvenile hormone and its related genes to DATFirst,we tested the protein content of the testis,ejaculatory duct,and male accessory glands of males after 7 hours of DAT fumigation.The results showed that the protein content of male accessory glands was significantly lower,the protein content of testeis was significantly higher,while the ejaculatory ducts protein contents were not different.Secondly,we also detected the change of male juvenile hormone titer after DAT fumigation for 7 hours.The results showed that the juvenile hormone titer was significantly higher than the controls,and then the juvenile hormone titer returned to the control level.In order to further explore the causes of juvenile hormone titer changes,we also measured the expression of related genes at different treatment times（fumigation 3hours,fumigation 7 hours,4 hours after fumigation,8 hours after fumigation,12 hours after fumigation）.The results showed that the Kr-h3 expression levers were consistent with the trend of juvenile hormone titer.The expression of the juvenile hormone response gene in fumigation was significantly higher after 7 hours fumigation than that in controls,and the expression at other time points was not significantly different from that in controls.The expression of the juvenile hormone synthetic gene MVk in five time points was significantly higher than that in control at 4 hours after fumigation,and the relative expression at other time points was not significantly different from that in controls.The expression of JHMAT in the first three time points was gradually higher than that in controls,and the expression of JHMAT at 4 hours after fumigation was significantly higher than that in controls.The expression of the juvenile hormone metabolic gene JHEH at fumigation 3 hours was significantly lower than that in controls,but the expression at other time points was not significantly different from that in controls.At the same time,Kr-h3,JHMAT,JHEH in different tissuses gene expression patterns also showed that the three genes are highly expressed in the testis.Finally,in order to verify the function of juvenile hormone,we tested the protein content of the testis,ejaculatory duct,and accessory glands of males after adding an exogenous juvenile hormone analogue（Methoprene）.The protein content of testis was significantly higher than the controls,but the content of accessory glands and ejaculatory duct was not significantly different from that of controls.2.Response of male ecdysone and its related genes to DATFirstly,we examined the changes of sperm count in testis,seminal vesicles and ejaculatory ducts after DAT fumigation for 7 hours.The results showed that sperm count in DAT treatment groups was significantly higher than that in controls after fumigation for 7 hours,but sperm count in double ejaculatory ducts was significantly lower than that in controls.At 4 hours after fumigation,sperm count in seminal vesicles and double ejaculatory ducts in DAT treatment group was significantly lower than that in controls.The results showed that in both groups the sperm count in the testis gradually decreases with time,while the sperm count in the seminal vesicles and double ejaculatory ducts gradually increases.But the change of testis in the DAT treatment group was always later than that in controls.This result indicated that DAT may possess the characteristic to decrease the releasing speed of sperm from the testis to the double ejaculatory duct,causing sperms to stay longer time in the testis,which finally resulted in the reduction of sperms in seminal vesicles and double ejaculatory ducts.Secondly,we measured the titer of ecdysone after fumigation for 7 hours.The results showed that the titer of ecdysone was significantly lower than the control level,then the titer of ecdysone returned to the control level.Further research found that the expression of male ecdysone-related genes at different treated times（fumigation 3 hours,fumigation 7 hours,4 hours after fumigation,8 hours after fumigation,12 hours after fumigation）also were all changed differently:at 4 hours after fumigation and 7 hours after fumigation,the expression of ecdysone responsive gene Ftz-f1 was significantly lower than that of controls;the expression of ecdysone synthesis gene Cyp302A1 after fumigation 3 hours was significantly lower than controls,while the expression of synthetic gene Cyp315A1 after fumigation 7 hours was significantly higher than that in controls,and the expression of synthetic gene Cyp314A1 at 4 hours after fumigation was significantly lower than that of controls.At the same time,the expression pattern in tissues of Ftz-f1 and Cyp302A1 was the highest in the double ejaculatory ducts of males.Finally,in order to verify the role of ecdysone in sperm release,we also measured the sperm count in the internal reproductive tissues and the expression of the Ftz-f1 gene after the treatment of ecdysone（20E）.The results showed that the sperm count in testis increased and the sperm count in double ejaculatory ducts decreased after 12 hours with treating 20E 10μg.At the same time,the expression of Ftz-f1 gene increased significantly after 12 hours and 24 hours with treating 10μg 20E.In conclusion,according to these results mentioned above,we speculated that DAT may disturb the corresponding hormone levels by affecting the expression of genes of juvenile hormone and ecdysone and then inhibit male reproduction.The above results provide a theoretical basis for the mechanism of diallyl trisulfide regulation of male S.cerealella reproduction.