| Reproduction and detoxification are two important biological processes requiring continuous energy supply.It is well known that the occurrence of insecticide resistance is usually concomitant with fitness cost related to insect reproduction,however,the underlying mechanisms remain largely unclear.AMP-activated protein kinase(AMPK)and Mammalian target of rapamycin/Ribosomal protein S6 kinase(mTOR/S6K)signaling pathways regulate the intracellular energy balance by sensing ATP and nutritional level,respectively,whereas the regulatory mechanisms of insect reproduction and detoxification by AMPK and S6K are still obscure.In this study,using Tribolium castaneum as a model insect,the temporal and spatial expression patterns of TcAMPKa and TcS6Kl and their responses of abiotic stresses were studied.In addition,the roles of TcAMPKa and TcS6K1 in regulating detoxification and reproduction as well as the underlying mechanisms were explored.1.Stress responses and functional characterization of AMPKαThe mRNA transcript,protein and phosphorylation levels of TcAMPKα under various environmental stresses including oxidative,heat(45℃)and cold(4℃)were quantified using RT-qPCR and western blot(WB).The results showed that the mRNA expression of TcAMPKa signifi cantly increased and reached a peak at 2h after paraquat treatment,which was 25.15fold higher than the control.The total protein level of TcAMPKa significantly increased by 3.33,2.35 and 2.95-fold at 2,4 and 12 h after paraquat treatment,respectively.The phosphorylation level of TcAMPKa Thr172 was 3.08-fold higher than control after paraquat treatment for 12 h.In addition,after 45℃ treatment for 4 h,the mRNA expression and phosphorylation levels of TcAMPKa reached the peak,which was 9.95-fold higher than the control.The mRNA expression level of TcAMPKa reached the peak after 4℃ treatment for 12 h,and the phosphorylation level of TcAMPKa reached the peak after 4 ℃ treatment 2 h,which was 1.76-fold higher than the control.Additionally,varying degree of upregulation of of TcAMPKa at the protein level was observed after heat and cold treatments for 1-12 h.The involvement of TcAMPKa in the tolerance to environmental stresses was further tested through RNAi.The results showed that,after exposure to oxidative stress-inducing 20 mM paraquat for 24 h,the mortality rate of dsTcAMPKa group was 69.04%,which was significantly higher than the WT(42.86%)and IB(38.10%)groups.Similarly,after heat and cold treatment,the survivorship of dsTcAMPKα-treated adults was much lower than WT and IB groups.The adults from dsTcAMPKa group could only survive for 26.00 h and 155.33 h at 45℃ and 4℃,respectively,which were much shorter than the WT group(55.00 h;242.67 h)and IB group(54.83 h;222.00 h).2.Metabolic and transcriptome responses of RNAi-mediated AMPKa knockdownThe role of TcAMPKa in lipid and carbohydrate metabolism was studied using RNAi.The TG level in dsTcAMPKa group was significantly increased by 53.49%when compared with the dsEGFP group.Similarly,increased glucose level by 62.34%was observed in the beetles injected with dsTcAMPKα when compared to the control beetles.However,the trehalose level in dsTcAMPKa group was significantly reduced by 8.56%compared with the dsEGFP group.Additionally,upon exposure to the AMPK activator aminoimidazole-4carboxamide1-β-D-ribofuranoside(AICAR),the levels of TG and glucose in AICAR group were significantly decreased by 34.60%and 41.89%,respectively,compared with injection buffer(IB)group,whereas the trehalose level increased by 17.07%in beetles treated with AICAR.Global unigene expression patterns of T.castaneum in dsTcAMPKα and dsEGFP groups were analyzed by RNA-Seq and RT-qPCR.A total of 1184 differentially expressed genes(DEGs)were obtained including genes involved in lipid and carbohydrate metabolism.The insect adipose triacylglycerol lipase homologue,brummer,which is responsible for the first step of TG hydrolysis,was significantly downregulated,whereas two fatty acid synthetase genes(FAS 1-2)involved in fatty acid biosynthetic pathways,and the transcription factor ChREBP,a key regulator of glucose and lipid metabolism and fat storage,were upregulated.To confirm the reliability of the DEG data,the expression levels of these DEGs were determined using RT-qPCR.Gene expression levels validated by RT-qPCR showed the high consistency with transcriptome sequencing.3.The pleiotropic AMPK-CncC signaling pathway regulates the trade-off between detoxification and reproductionThe effects of deltamethrin treatment on the mRNA transcript,protein and phosphorylation levels of TcAMPKa were detected by RT-qPCR and Western Blotting.The results showed that both mRNA and phosphorylation levels of TcAMPKα were induced by deltamethrin treatment in T.castaneum female adults with the peak observed at 4 h after treatment when compared with the controls.In addition,deltamethrin treatment upregulated total-TcAMPKa protein level with the peak observed at 2 h after treatment.The MDA,ATP,ADP and AMP levels after exposure to deltamethrin were detected by ELISA.The results showed that the content of MDA reached the highest level at 12 h after treatment,which was 2.47-fold higher than the control.In addition,the AMP:ATP and ADP:ATP ratios were significantly enhanced after deltamethrin treatment for 1 to 12 hours.The effects of dsTcAMPKα and/or AICAR treatment on deltamethrin tolerance and mRNA expression of CYP6BQs regulated by transcription factor Cap ’n’ collar isoform C(CncC)were detected by bioassay and RT-qPCR.After exposure to deltamethrin for 24 h,the survival rate of dsTcAMPKα-injected beetles was 23.33%,which were significantly lower than the dsEGFP group(50%).The injection of dsTcAMPKα attenuated the induction of CncC target genes,CYP6BQs by deltamethrin.On the contrary,AICAR treatment upregulated the expression of CYP6BQ genes.Further experiments showed that AICAR treatment significantly decreased number of eggs laid per female by 94.06%and juvenile hormone(JH)levels compared with controls,whereas increased the ecdysteroid contents.Accordingly,RTqPCR showed that mRNA levels of two ecdysteroid biosynthesis genes,TcPhantom and TcShade,the ecdysone receptor gene TcEcR and TcUSP,as well as the JH degradation genes JHEH-r3 and JHEH-r4 were induced by AICAR treatment,whereas the expression of JH early-response gene Kr-h1(Kruppel homolog 1)and JH receptor Met(Methoprene tolerant)were significantly decreased at 48 h after AICAR treatment.In contrast,there is no significant difference in the expression of gene coding for the key enzyme that acts at the final steps of the JH biosynthesis pathway,JHA methyltransferase(JHAMT).The interaction of TcAMPKa and TcCncC in vivo or in vitro was revealed by using immunofluorescence,yeast two hybrid,co-immunoprecipitation,GST pull-down and bimolecular fluorescent complimentary.Subcellular localization analysis showed that deltamethrin treatment induced the nuclear accumulation of TcCncC and the accumulation was attenuated by dsTcAMPKα injection after deltamethrin treatment for 4 h.Furthermore,detection of phosphorylation level by Phosphorylation level detection kit showed that TcAMPK-αβγ T172D recombinant protein promoted phosphorylation level of TcCncC.WB detection found that AICAR and deltamethrin treatment could induce the immunoprecipitated TcCncC Ser/Thr phosphorylation levels,whereas the dsTcAMPKa treatment could inhibit the deltamethrin-induced phosphorylation.The roles of TcCncC in the regulation of genes involved in JH and 20-hydroxyecdysone(20E)signaling pathways were detected by RNAi and RT-qPCR.Knockdown of TcCncC significantly decreased expression of genes involved in ecdysteroid biosynthesis(TcPhantom and TcShade)and action(TcEcR and TcUSP)as well as JH degradation(TcJHEH-r2,TcJHEHr3,TcJHEH-r4)In contrast,increased expression levels of genes involved in JH action including TcKr-h1(Kruppel homolog 1)and TcMet were observed in dsTcCncC-injected beetles compared with dsEGFP-injected beetles.Co-expressed TcCncC and its partner protein TcMaf could enhance the luciferase activity of TcPhantom,TcShade and TcJHEHr3 promoters.The results of electrophoretic mobility shift assay(EMSA)showed that TcCncC could bind to the conserved binding site in the TcPhantom,TcShade and TcJHEHr3 promoters.4.Regulatory role of TcS6Kl in reproductionThe roles of TcS6K1 in reproduction and its effects on the mRNA expression of JH biosynthesis-and reproduction-related genes as well as transcription factor Forkhead box O(FOXO)were studied by RNAi and RT-qPCR.The results showed that the number of eggs each pair per day in both FemaleTcS6K1RNAi × MaleWT and FemaleWT × MaleTcS6K1 RNAi groups were dramatically lower than FemaleIB × MaleWT and FemaleWT × MaleIB groups,respectively.The dsTcS6K1 injection in both females and males led to the down-regulation of JH biosynthesis related gene TcFDH compared with IB groups.In addition,the mRNA expression of TcVgs were also reduced in dsTcS6K1 injected female beetles,and the mRNA expression of two accessory gland secretory genes in dsTcS6K1 injected male beetles were downregulated compared with controls.The effect of dsTcS6K1 treatment on the subcellular localization of TcFOXO as well as the transcriptional regulation of JH biosynthesis-related genes by TcFOXO were studied by RNAi,WB and EMSA.dsTcS6K1 treatment promoted the TcFOXO nuclear accumulation,whereas the total protein level of TcFOXO was not affected.Knockdown of TcFOXO dramatically increased the TcFDH mRNA expression level by 7.74 times.However,dsTcFOXO treatment had no effects on the expression of TcJHAMT and TcS6K1.Further EMSA assay showed that the purified TcFOXO protein could bind to biotin-labeled probe covering the conserved FOXO binding motif in the promoter of TcFDH.5.The role and underlying mechanisms of TcS6K1 in detoxificationThe effects of deltamethrin treatment on mRNA,total protein and phosphorylation levels of TcS6K1 were detected by RT-qPCR and WB.Varying degree of downregulation of TcS6K1 at the mRNA level was observed after deltamethrin treatment for 1-12 h,and the lowest mRNA levels of TcS6K1 was observed at 2 h after deltamethrin treatment,which was decreased by 97.88%compared with control.Gray value analysis showed that the total protein expression level of TcS6K1 was down-regulated by 42.76%after deltamethrin treatment for 12 h.The phosphorylation level of TcS6K1 Thr389 was up-regulated by 1.28 times after deltamethrin treatment for 4 h.The effects of dsTcS6K1 and MHY1485(S6K1 activator)treatment on deltamethrin tolerance and related antioxidant enzyme activities were detected by bioassay and ELISA.Knock down of TcS6K1 by RNAi decreased the sensitivity of T.castaneum to deltamethrin.The survival rate under deltamethrin treatment was up-regulated by 33.33%in dsTcS6K1injected larvae compared with control,whereas the survival rate was down-regulated by 26.67%upon the activation of S6K1 by MHY1485.Similarly,the activities of GST,CAT and SOD were upregulated by 1.36,4.06 and 1.72 times,respectively,in dsTcS6K1-injected larvae compared with controls.Conversely,after MHY1485 treatment,the activities of,GST,CAT and SOD were downregulated by 42.23%,42.92%and 90.93%,respectively,compared with controls.The interactions between TcS6K1 and TcAMPKa were explored by immunofluorescence,RNAi and WB,Midguts were dissected from deltamethrin-treated larvae and stained with antiTcS6K1 and anti-TcAMPKa specific antibodies for co-localization detection.The results showed that TcS6K1 protein was co-localized with TcAMPKa protein and the overlap coefficient was approaching to 100%.RNAi of TcS6K1 had not effect on TcAMPKa mRNA and protein expression levels,but significantly increased the phosphorylation level of TcAMPKa by 3.71 times compared with control.In addition,knockdown of TcS6K1 increased the AMP/ATP and ADP/ATP ratios by 1.93 and 2.14 times,respectively.iTRAQ-based quantitative proteomic analysis was conducted to explore the overall protein expression patterns in dsTcS6K1一and dsEGFP-treated larvae and a total of 1184 differentially expressed proteins(DEPs)were identified including proteins involved in detoxification,antioxidant and ATP biosynthesis/hydrolysis.RT-qPCR was further conducted,and the results were generally consistent with proteomic results.The upregulation of detoxification-related DEPs contained Glutathione S-transferase theta-1(GSTtl),Venom carboxylesterase-6(VCE6),Cytochrome P450 9e2(CYP9e2)and Cytochrome P450 4C1-like(CYP4C1).The up-regulation of antioxidant-related DEPs contained catalase-like(CAT)and superoxide dismutase[Mn](MnSOD).In addition,the ATP biosynthesis-related ATP synthase subunit s-like protein(ATP5SL)was down-regulated,whereas ATP hydrolysis-related ATPase N2B(HFN2B)was up-regulated.RT-qPCR results showed that deltamethrin treatment significantly increased the TcCYP9e2 and TcVCE mRNA expression in dsEGFP-injected larvae compared with the control,whereas the TcCYP4Cl mRNA expression was not affected.In addition,TcCYP9e2,TcCYP4Cl and Tc VCE mRNA expression levels in dsTcS6K1-injected larvae were 13.73,4.96 and 1.32-fold higher,respectively,than in dsEGFP-injected larvae under deltamethrin treatment.Further analysis with RNAi found that the survival rate of dsTcCYP9e2-injected larvae under deltamethrin treatment was significantly downregulated by 41.67%compared with control.Taken together,the results in this study indicated that TcAMPKa could protect T.castaneum from harmful environmental factors and oxidative damage and transcriptionally regulate the genes involved in lipid and carbohydrate metabolism.The pleiotropic AMPKCncC signaling could mediate the trade-off between detoxification and reproduction by regulating transcr-iption of related genes.TcS6K1 could regulate the reproduction of T.castaneum through FOXO-JH signaling pathway,and regulate the TcAMPK activity by regulating expression of ATP biosynthesis-as well as hydrolysis-related genes.Additionally,TcS6K1 mediated the sensitivity of T.castaneum to deltamethrin by regulating the expression of detoxification-and antioxidant-related genes.These results have important scientific significance for further revealing the functions of AMPK and S6K1 and the trade-off mechanism of detoxification and reproduction in insects. |
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