Regulation Mechanism Of Infertility Induced By Azadirachtin In Spodoptera Litura(Fabricius)

As a typical representative of modern botanical insecticides,azadirachtin has extensive development potential and application prospects due to its obviously biological activities for insects,such as antifeedant,repellent,inhibit growth and infertility.Therefore,azadirachtin can be used to regulate insect reproduction in production practice,and the population of insects will be controlled below the economic threshold,which can provide an important practical reference for achieving green prevention and control,however,at present,the regulation mechanism of insects infertility induced by azadirachtin is still not clear.Spodoptera litura(Fabricius)is a mainly lepidopteran pest with strong fertility.In this study,the effects of azadirachtin on the growth and development and fertility of S.litura are determined.To further explore the molecular mechanism,iTRAQ(isobaric tags for relative and absolute quantitation)based approach is applied to identify the differentially expressed proteins(DEPs)regulated by azadirachtin in testis and ovary,respectively.Based the results from iTRAQ and function analisis for DEPs,the mechanism of azadirachtin regulating the infertility was explored in S.litura.The main results are as follows:(1)To assess the effects of azadirachtin on male fertility,the copulation process was conducted by using treated mature male adult flies(treated by different doses of azadirachtin,0.05,0.1 and 0.15 mg/L,respectively)to mate with untreated female flies at the same stage.The bioactivity assay results indicated that the actual numbers of eggs,oviposition duration and oviposition peak duration were all significantly reduced after male moths were exposed to azadirachtin,and the effect was increased as the increasing concentration of azadirachtin,the inhibition rate for the actual numbers of eggs were 32.33%,78.76% and 100%,respectively.(2)iTRAQ-based proteomic was used to analysis the mechanism of male infertility induced by azadirachtin in male S.litura.The insects were treated by 0.1 mg/L azadirachtin from third instar larvae till pupation and emergence,the protein changes of testis from pupae(7th day after hatch)and adults(2nd day after emergence)were identified based on a 1.2-fold change threshold compared with un-treated group.The results suggested that a total of 275 and 134 DEPs were identified at pupae and adult stage,respectively.The results from KEGG analysis suggested that DEPs at pupae stage were mainly enriched in the pathways involved in apoptosis,including Focal adhesion,while those at adult stage were mainly enriched in the pathways involved in metabolism process,including AMPK signaling pathway.(3)To verify the occurrence of azadirachtin-induced apoptosis at different stages(pupae and adult),the ultrastructure of cells,TUNEL-positive cells and the caspase-3 enzymes activity and the expression of caspase-3 in testis at two developmental stages were analyzed.The results suggested that azadirachtin at the concentration of 0.1 mg/L can up-regulate the expression of caspase-3 at both m RNA and protein levels and increase the activity of caspase-3 in cells at pupae stage.TUNEL analysis showed that azadirachtin can induce the presence of FITC-d UPlabeled green fluorescent signal in the testis cells at pupae stage,but TUNEL-positive cells were not observed at adult stage.By TEM analysis,the results showed that cell chromatin condensation,cell volume contraction were observed at pupae stage,but not in adult stage.These results consistently indicated that azadirachtin can induce apoptosis in testis of S.litura at pupae stage,which may be an important reason for the weakening of female fecundity after treated mature male adult flies to mate with untreated female flies.(4)To assess the effects of azadirachtin on female fertility,the copulation process was conducted by using treated mature female adult flies(treated by different doses of azadirachtin,0.05,0.1 and 0.15 mg/L)to mate with untreated male flies at the same stage.The bioactivity assay results showed that the actual numbers of eggs,total numbers of eggs,oviposition duration and oviposition peak duration all significantly reduced after male moths were exposed to azadirachtin,and the effect was increased as the increasing concentration of azadirachtin,the inhibition rate for actual numbers of eggs were 54.07%,80.94% and 100% at the concentration of azadirachtin were 0.05,0.1 and 0.15 mg/L,respectively;and the inhibition rate for total numbers of eggs were 42.23%,77.73% and 96.72% at the concentration of azadirachtin,respectively.(5)iTRAQ-based proteomic was used to analysis the mechanism of infertility induced by azadirachtin in female S.litura.The insects were treated by 0.1 mg/L azadirachtin from third instar larvae till pupation and emergence,the protein changes of ovary from pupae(7th day after hatch)and adults(2nd day after emergence)were identified based on a 1.2-fold change threshold compared with un-treated group.The results suggested that a total of 919 and 530 DEPs were identified at pupae and adult stage,respectively.The results from KEGG analysis suggest that DEPs in pupae were mainly enriched in the pathways involved in metabolism,including carbon metabolism,the differentially accumulated proteins in adult were mainly enriched in the pathways involved in endoplasmic reticulum stress(ERS)response and lipid metabolism.(6)Biochemical analysis and TEM analysis were carried out to verify endoplasmic reticulum stress could be induced by azadirachtin 0.1 mg/L in ovary of adult flies.The results showed that azadirachtin increased the concentration of ROS with 2.22-fold change.The TEM analysis observed large number of degenerated yolk granules and swollen endoplasmic reticulum were accumulated in the ovary cell of adult flies following the larva were treated from third instar.(7)Western blot was carried out to analysis the role of unfold protein response(UPR)during the ERS induced by azadirachtin at the concentration of 0.1 mg/L.The results suggested that azadirachtin could active the ATF6 signaling pathway by increasing the protein expression of ATF6;however,azadirachtin could inhibite the PERK signaling pathway by reducing the protein expression of PERK,P-PERK and P-EIF2;and azadirachtin could inhibite the IRE1-JNK signaling pathway and induced the apoptosis by reducing the protein expression of IRE1,P-IRE1,IRS1,P-JNK,Bcl2,P-IRE1 and increasing the protein expression of JNK and INS.(8)To verify the occurrence of azadirachtin-induced apoptosis at different stages(pupae and adult),the expression of caspase-3 in ovary and the caspase-3 enzymes activity at two developmental stages were analyzed.The results showed that azadirachtin at the concentration of 0.1 mg/L can up-regulate the expression of caspase-3 at m RNA and protein levels and increase the activity of caspase-3 in cells.At pupae stage,western blot analysis indicated that the expression of AKT,P-AKT,Pi3 K and p-m TOR were significantly up-regulated after azadirachtin treatment.However,at adult stage,the expression of P-AKT、Pi3K and p-m TOR were significantly down-regulated after azadirachtin treatment,while the expression of AKT was up-regulated.The results above suggested that the concentration of 0.1 mg/L can induce apoptosis mediated by PI3K/AKT pathway in ovary at adult stage.Besides,by using HE and TUNEL analysis,it was summarized that azadirachtin can cause pathological changes and apoptosis of ovarian tube tissues,which mainly occured in nurse cells and follicular cells in the vitellogenesis area of ovarian tubes.(9)To analysis the effects of azadirachtin on lipid metabolism of ovary,the insects were treated by 0.1 mg/L azadirachtin from third instar larvae till pupation and emergence.The ovary was selected from the female adult at the 2nd day of emergence.Biochemical analysis showed that azadirachtin could increase the content of insulin in ovary with 1.92-fold changes and reduce the content of triglycerides(TG)and glycogen(Gly)with 57.74% and 57.77%-fold changes,respectively.The results of q RT-PCR suggested that compared with the control groups,the m RNA expression level of FASN and ACACA were decreased significantly,the expression level of SCD no significant difference.Western blot showed that the proteins expression level of FASN,ACACA and P-ACC were decreased obviously,while the expression level of SCD no significant difference.Besides,it was found that the ability of synthesize new proteins in endoplasmic reticulum were inhibitd after azadirachtin treatment by using SUn SET assay.The results from ELISA assay showed that azadirachtin reduced the concentration of vitellogenin(VTG)and vitellin(Vn).These results indicated that azadirachtin could inhibit ovarian development at adult stage by regulating lipid metabolism at a concentration of 0.1 mg/L.Based on the above results,azadirachtin could significantly inhibit the development of ovary of S.litura,and the inhibition rates for the length and weight of ovary were 49.45% and 58.80%,respectively.These results indicated that azadirachtin may break the endoplasmic reticulum homeostasis in ovarian tissue cells and induce apoptosis,which inhibited the lipid metabolism and protein synthesis of ovary,and might be an important reason for attenuating the fertility in female S.litura.