Physiological mitochondrial ROS regulate diapause by enhancing HSP60/Lon complex stability in Helicoverpa armigera

ZHANG Xiao-shuai,SU Xiao-long,GENG Shao-lei,WANG Zheng-hao

School of Life Sciences,Sun Yat-Sen University,Guangzhou 510006,P.R.China

Abstract Diapause is a long-lived stage which has evolved into an important strategy for insects to circumvent extreme environments.In the pupal stage,Helicoverpa armigera can enter diapause,a state characterized by significantly decreased metabolic activity and enhanced stress resistance,to survive cold winters. Previous studies have shown that reactive oxygen species (ROS) can promote the diapause process by regulating a distinct insulin signaling pathway. However,the source of ROS in the diapause-destined pupal brains and mechanisms by which ROS regulate diapause are still unknown. In this study,we showed that diapause-destined pupal brains accumulated high levels of mitochondrial ROS (mtROS) and total ROS during the diapause process,suggesting that mitochondria are the main source of ROS in diapause-destined pupal brains. In addition,injection of 2-deoxy-D-glucose (DOG),a glucose metabolism inhibitor,could delay pupal development by elevating mtROS levels in the nondiapause-destined pupal brains. Furthermore,the injection of a metabolite mixture to increase metabolic activity could avert the diapause process in diapause-destined pupae by decreasing mtROS levels.We also found that ROS could activate HSP60 expression and promote the stability of the HSP60-Lon complex,increasing its ability to degrade mitochondrial transcription factor A (TFAM) and decreasing mitochondrial activity or biogenesis under oxidative stress. Thus,this study illustrated the beneficial function of ROS in diapause or lifespan extension by decreasing mitochondrial activity.

Keywords:mtROS,diapause,HSP60,Helicoverpa armigera

1.Introduction

As the largest group of species on earth (Zhang 2011),insects can enter diapause in a specific stage during exposure to unfavorable environments. Diapause,akin to dauer inCaenorhabditiselegans,is a long-lived “non-aging”stage,because it persists for months and dramatically extends lifespan (Hu 2007;Luet al.2013). Diapause can occur in a variety of developmental stages,such as the embryonic,larval,pupal and adult stages. For example,the cotton bollworm,Helicoverpaarmigera,can enter diapause at the pupal stage,which is characterized by significantly decreased metabolic activity and enhanced stress resistance,to survive cold winters.

Mitochondrial reactive oxygen species (mtROS) are generally associated with ageing (Balabanet al.2005;Halliwell 2006). A high concentration of mtROS can damage proteins,lipids,and DNA and is associated with numerous diseases,such as cancer and neuro-degenerative disorders(Balabanet al.2005;Halliwell 2006;Wellen and Thompson 2010). However,pioneering works inC.eleganshave shown that mildly increased mtROS due to mutations of mitochondrial genes or chemical inhibition that affect mitochondrial function can lengthen rather than shorten the lifespan (Schulzet al.2007;Yang and Hekimi 2010). Such effects are observed in many species other thanC.elegans.For example,mutation of target of rapamycin 1 in yeastSaccharomycescerevisiae(Panet al.2011) and allotopic expression of fungal NADH dehydrogenase in theDrosophila melanogasterbrain (Scialoet al.2016) also prolong lifespan in a reactive oxygen species (ROS)-dependent manner.Interestingly,a previous report indicates that ROS levels are significantly higher in diapause-destined pupal brains than in brains from nondiapause-destined pupae (Zhanget al.2017). The mildly increased ROS can extend the lifespan in the pupae ofH.armigeraat natural physiological levels by regulating the insulin signaling pathway. However,the source of ROS in the diapause-destined pupal brains and molecular mechanisms underlying the effects of ROS on diapause are still unclear.

Under oxidative stress conditions,proteins are at risk of inactivation by misfolding,unfolding,or aggregation. The mitochondrial chaperonin heat shock protein 60 (HSP60)primarily facilitates protein folding and prevents unfolded protein aggregation (Chenget al.1989;Fanet al.2020).HSP60 has been shown to closely cooperate with ATPdependent proteases to recognize and remove dam aged proteins in mitochondria under oxidative stress (Benderet al.2011). The mitochondrial Lon protease,which is well conserved among species,is a member of the superfamily of ATPases associated with diverse cellular activities,and plays a role in protein quality control surveillance in mitochondria by degrading preferentially oxidized or misfolded proteins (Van Melderen and Aertsen 2009;Voos 2009;Matsushimaet al.2010;Bota and Davies 2016).Mitochondrial transcription factor A (TFAM),a nuclearencoded high-mobility group (HMG) family protein,is the essential component of the mitochondrial nucleoid core complex (Canugoviet al.2010). TFAM has been reported to function as a mitochondrial transcription factor for regulating the mitochondrial activity and biogenesis by binding to the mitochondrial light strand promoter (Kanget al.2007). The down-regulation of TFAM has been reported to decrease mitochondrial activity and biogenesis,and ultimately regulate pupal diapause inH.armigera(Linet al.2016).However,the molecular mechanism of ROS-controlled HSP60 and Lon activity in insect diapause is unclear.

Here we showed that during the diapause process inH.armigera,the pupal brains accumulated high levels of mtROS and total ROS. Furthermore,we showed that high levels of mtROS were achieved in response to decreased glucose utilization,because injection of 2-deoxy-D-glucose(DOG),a glucose metabolism inhibitor,could elevate mtROS levels in the nondiapause-destined pupal brains and delay pupal development. On the other hand,the injection of a metabolite mixture which includes glucose,pyruvate,malic acid,and fumaric acid,decreased the mtROS levels in the diapause-destined pupal brains and averted the diapause process. Furthermore,HSP60,which was located in mitochondria and up-regulated by ROS,could bind to Lon and the stability of this complex was enhanced by ROS. Activated Lon promoted the degradation of oxidized mitochondrial proteins,such as TFAM,which led to decreased mitochondrial activity and biogenesis.

2.Materials and methods

2.1.Insects

Helicoverpa armigeralarvae were reared on an artificial diet at (20±1)°C. To produce nondiapause type pupae,the light-dark cycle was 14 h:10 h;for diapause type pupae,the cycle was 10 h:14 h. All the nondiapause-destined pupae developed without entering diapause;and the diapause rate was over 95%. The new pupae were collected for synchronizing the developmental stages as previously described (Zhanget al.2017).

2.2.Measurement of mtROS and total ROS content

Measurement of mtROS was performed according to the method of Mukhopadhyayet al.(2007) with modifications.Briefly,pupae were injected with the indicated treatment.After treatment,15 pupal brains were dissected as a sample and immediately stored in liquid nitrogen. Three samples were tested at each point. The brains were digested with 400 μL trypsin (Sigma,USA) for 10 min and 40 μL fetal bovine serum was added for digestion termination.Trypsinized cells were pelleted at 12 000×g (4°C) for 5 min and then re-suspended in PBS buffer containing 2 μmol L-1MitoSOX for 30 min at room temperature. After incubation,the cells were washed three times with PBS and then re-suspended in 400 μL PBS. Fluorescence intensity was analyzed by flow cytometry with excitation/emission maxima of approximately 488/565 nm. The unit of mtROS is indicated by the mean fluorescence intensity of MitoSOX.

The measurement of total ROS in the brains was performed as previously described (Zhanget al.2017).Briefly,pupal brains were collected and immediately stored at -80°C. The brain extracts were prepared by sonication in NP40 buffer and centrifuged at 12 000×g (4°C) for 20 min,and the supernatant was then used for protein quantification by the Bradford assay. Supernatant containing 10 μg of protein was then pre-incubated with 10 μmol L-1of H2DCFDA (Invitrogen,USA) in 100 μL of PBS at 37°C for 1 h.Fluorescence intensity was assayed with a fluorimeter with the excitation wavelength 485 nm and the emission wavelength 535 nm. The unit of total ROS is indicated by the fluorescence arbitrary unit of H2DCFDA.

2.3.Effects of injection of DOG,N-acetyl-L-cysteine(NAC),or metabolite mixture on H.armigera development

Diapausing and nondiapausing pupae were assessed as reported in Phillips and Newsom (1966). In nondiapause pupae,the stemmata gradually migrate until they disappear,however,in diapause pupae the stemmata will always remain in a straight line.

Day 1 nondiapause-destined pupae were either injected with 3 μL of 2 mmol L-1DOG or injected with 450 μg NAC after the injection of an equal amount of DOG in 1 h,and in both treatments the pupae were then incubated at 22.5°C.Pupal developmental delay was determined by examining the pupal stemmata location on different days after injection.

Day 1 diapause-destined pupae were injected with metabolite mixture and incubated at 22.5°C. The dose of injected metabolite mixture contained 150 μg glucose,120 μg pyruvate sodium,300 μg malate and 20 μg fumarate.Pupal development was determined on day 10 after injection by determining the pupal stemmata location.

2.4.RNA extraction and DNA amplification

Pupal brain total RNA was extracted by using Trizol as previously described (Chen and Xu 2014). Briefly,total RNA(1 μg) was reverse transcribed using an M-MLV Reverse Transcription System (TaKaRa,Japan) at 37°C for 1 h. The reverse transcription product (1 μL) was added to 50 μL of the PCR reaction system,and full length amplification of HSP60 was performed with the specific primers shown in Appendix A.

2.5.Cell culture and transfection

HzAm1 cells were cultured at 27°C in Grace’s insect cell culture with 10% fetal bovine serum. Cells were transfected using the FuGENE?HD Transfection Reagent (Promega,USA) according to the manufacturer’s instructions. Briefly,cells were suspended and plated in a glass bottom dish (for the HSP60 and mitochondria co-localization assay) or 6-well plates (for the immunoprecipitation assay) and cultured for 12 h. Firstly,sterile water was added to a sterile tube so that the final volume after adding the transfection reagent/DNA or dsRNA mixtures was 100 μL. GFP-HSP60 plasmids (1 μg)or HSP60 dsRNA (4 μg) were then mixed with transfection reagent in a ratio of 1:3 in the sterile tube. The FuGENE?HD Transfection Reagent/DNA or dsRNA mixtures were incubated at room temperature for 20 min,and added to the wells. The plates were gently shaken and then returned to the incubator for the indicated time.

2.6.HSP60 and mitochondria co-localization

HzAm1 cells were plated in a glass bottom dish (Nunc,Danmark) 12 h prior to transfection. At 36 h posttransfection with the GFP-HSP60 plasmids,mitochondria were stained with Mito-tracker red (Invitrogen) for 10 min,as recommended by the manufacturer,prior to cell washes with PBS. Nuclei were stained with Hoechst dye (Sigma) for 10 min,and washed cells with PBS. Images were obtained with a Leica confocal upright microscope.

2.7.Polyclonal antibody preparation

Har-Actin and Har-HSP60 polyclonal antibody preparations were performed as previously described (Zhanget al.2017).Briefly,partial sequences of Har-Actin and Har-HSP60 were cloned into the pET32a vector (Invitrogen). The recombinant proteins of Har-HSP60 and Har-Actin were expressed in BL21 (DE3) by induction with IPTG at 20°C for 9-12 h,and then purified on a NTA-Ni2+-agarose column (Qiagen,China). Purified proteins were assayed using the Bradford assay and then used to prepare polyclonal antibodies in rabbits (Bradford 1976;Chen and Xu 2014;Liet al.2018).

2.8.Protein extraction and Western blot

Protein extraction and Western blot analysis were performed as previously described (Zhanget al.2017). The pupal brains or brain-SG complexes were lysed in NP40 buffer (1% NP-40,150 mmol L-1NaCl,50 mmol L-1Tris-HCl (pH 8.0),0.1%SDS,0.5% sodium deoxycholate,1 mmol L-1EGTA,1 mmol L-1PMSF,5 mmol L-1NaF,and 10 mmol L-1Na3VO4). The extracts were shaken in a rotary shaker at 4°C for 1 h,followed by centrifugation at 12 000×g at 4°C for 20 min.

Proteins were separated on a 10% SDS-PAGE gel,and then transferred to PVDF membranes. The immunoreactivity was detected with antibodies against HSP60,Lon (Affinit,DF12119),TFAM,and Actin. The dilutions of the HSP60 and TFAM,Lon and Actin antibodies were 1:3 000,1:1 000;and 1:10 000,respectively. The dilutions of secondary antibodies were at 1:3 000-1:10 000. Immobilon Western Chemiluminescent HRP Substrate (Thermo,USA) was used for protein detection. The specificities of the HSP60,Lon,and TFAM antibodies are shown in Appendix B.

2.9.Co-immunoprecipitation analysis

Pupal brains were lysed in NP-40 cell lysis buffer and protein extract (500 μg) was applied for the co-immunoprecipitation assay. The co-immunoprecipitation systems contained 35 μL Protein G/A plus-agarose suspensions (Merck,USA) and 1 μg antibody. As a negative control,the same amount of IgG (normal rabbit serum) was used instead of the antibodies. Immunoblotting was performed with the corresponding antibodies,followed by secondary antibody incubation with Clean-blot HRP (Thermo,USA) at a dilution of 1:1 000,and then the blot was detected.

To test whether HSP60 regulates the combination of Lon and TFAM under oxidative stress,we carried out HSP60 RNA interference in HzAm1 cells. Briefly,HzAm1 cells were transfected with 4 μg HSP60 dsRNA for 48 h,and then treated with 200 μmol L-1H2O2for 30 min. Protein extract (500 μg) was applied for the co-immunoprecipitation assay. The co-immunoprecipitation systems contained 35 μL Protein G/A plus-agarose suspensions (Merck,USA)and 1 μg Lon antibody,followed by immunoblotting with HSP60 and TFAM antibodies,respectively.

2.10.Statistical analysis

All statistical analyses were performed with SPSS 19.0(IBM Corporation,New York,USA). Student’st-test was used to analyze the significance of difference. Error bars represent SD.

3.Results

3.1.High levels of physiological mtROS and total ROS accumulate in the diapause-destined pupal brains of H.armigera

Using the specific fluorescent probe MitoSOX,we investigated the levels of mtROS in pupal brains of diapause-and nondiapause-destined individuals. As shown in Fig.1-A,mtROS levels in diapause-destined pupal brains were significantly higher than in nondiapause brains from day 0 to day 15. In addition,the total ROS levels detected by H2DCFDA were significantly higher in diapause-destined pupal brains than in nondiapause-destined pupal brains (Fig.1-B).

Fig.1 Developmental changes in mitochondrial reactive oxygen species (mtROS) in the diapause-destined and nondiapausedestined pupal brains. A,changes of mtROS in the pupal brains. mtROS was detected using MitoSOX and flow cytometry.B,changes of total ROS in the pupal brains. Total ROS was detected using H2DCFDA. DP,diapause-destined pupae;NP,nondiapause-destined pupae;F.A.U.,fluorescence arbitrary unit. Values are expressed as mean±SD (n=3). ＊,P＜0.05;＊＊,P＜0.01(Student’s t-test).

We also determined the superoxide dismutase (SOD)and catalase activities,and found that the activities of both were significantly lower in diapause-destined pupal brains than in nondiapause-destined pupal brains (data not shown).

3.2.High physiological mtROS delay development in response to decreased glucose utilization

To clarify the mechanism of mtROS in the regulation of insect diapause,DOG was injected with or without NAC(a ROS scavenger) into day 1 nondiapause-destined pupae and the subsequent timing of the migration of the pupal stemmata was monitored. In controls injected with solvent,the stemmata of 50% of the individuals migrated in 3 days,whereas stemmata migration was delayed by 1.5 days by injection of DOG (Fig.2-A). On the contrary,the development-delaying effect of DOG injection was significantly rescued by the injection of NAC. Furthermore,after injection of DOG into day 1 nondiapause-destined pupae,mtROS was significantly elevated in a dosedependent manner (Fig.2-B). However,we then injected a metabolite mixture that included glucose,pyruvate,malic acid,and fumaric acid,into day 1 diapause-destined pupae to increase metabolic activity,and found that the diapause process was averted,and the pupae proceeded with nondiapause development (Fig.2-C). Injection of the metabolite mixture also significantly decreased the mtROS levels in the pupal brains in comparison to the control(Fig.2-D). These findings suggested that mtROS play an important role in diapause.

Fig.2 Mitochondrial reactive oxygen species (mtROS) promotion of diapause in response to decreased glucose utilization. A,decreased glucose utilization by injection of 2-deoxy-D-glucose (DOG) delays the stemmata migration in an ROS-dependent manner. Day 1 nondiapause-destined pupae were injected with the indicated compounds of H2O (n=35),DOG (n=35) and DOG+N-acetyl-L-cysteine (NAC) (n=26). B,changes of mtROS in response to decreased glucose utilization in the brains. Day 1 nondiapause-destined pupae were injected with 3 μL DOG solutions for 48 h (n=3). C,increased glucose metabolism by injection of metabolite mixture promotes the stemmata migration. Day 1 diapause-destined pupae were injected with metabolite mixture and incubated at 22.5°C (n=67 for no-treat;n=61 for H2O;n=59 for metabolite mixture). D,changes of mtROS in response to increased glucose metabolism in the brains. Day 1 diapause-destined pupae were injected with metabolite mixture for 96 h (n=3).Values are expressed as mean±SD. ＊,P＜0.05;＊＊,P＜0.01 (Student’s t-test).

3.3.mtROS up-regulate mitochondrial HSP60 expression

To test the hypothesis of a role for mtROS in regulating diapause,we subsequently investigated the expression of HSP60,a mitochondrial heat shock protein,by Western blot analysis.The expression of HSP60 in diapausedestined pupae was significantly higher from day 0 to day 4 than in nondiapause-destined pupae of the same stage(Fig.3-A). In addition,we detected the localization of HSP60 in mitochondria. As shown in Fig.3-B,HSP60 was co-localized with mitochondria that had been stained by Mito-Tracker. These results suggested that HSP60 functions as a mitochondrial chaperone in diapause initiation.

To assay the HSP60 function in insect diapause,we injected DOG into day 1 nondiapause-destined pupae.Western blot results showed that HSP60 expression significantly increased in response to ROS (Fig.3-C). We then injected antimycin A,a compound which increases mtROS generation (Westet al.2011;Duttaet al.2013),into nondiapause-destined pupae to specifically increase mtROS in pupal brain. The results showed that HSP60 expression also significantly increased when antimycin A was injected into day 1 nondiapause pupae (Fig.3-D).Furthermore,NAC,as a ROS scavenger,was injected into day 1 diapause-destined pupae,and HSP60 expression significantly decreased in comparison to the control(Fig.3-E).

Fig.3 Mitochondrial HSP60 activation by mitochondrial reactive oxygen species (mtROS). A,changes in the expression of HSP60 in the pupal brain. DP,diapause-destined pupae;NP,nondiapause-destined pupae. B,HSP60 localization in mitochondria. C,effects of 2-deoxy-D-glucose (DOG) on HSP60 expression. Day 1 nondiapause-destined pupae were injected with 3 μL DOG solutions for 48 h,and protein was extracted from brains for Western blot analysis. D,effects of antimycin a (AA) on HSP60 expression. Day 1 nondiapause-destined pupae were injected with 0,7.5,15 and 30 ng AA for 4 h,and protein was extracted from brains for Western blot analysis. E,effects of N-acetyl-L-cysteine (NAC) on HSP60 expression. Day 1 diapause-destined pupae were injected with the indicated dose of NAC for 48 h,and protein was extracted from brains for Western blot analysis. Values are expressed as mean±SD (n=3). ＊,P＜0.05;＊＊,P＜0.01 (Student’s t-test).

3.4.ROS/HSP60 regulates mitochondrial activity by enhancing the binding of Lon with TFAM

To functionally evaluate the role of HSP60 in diapause in response to mtROS,Lon and TFAM were analyzed. First,a co-immunoprecipitation assay showed that HSP60 could combine with Lon to form a complex (Fig.4-A). Furthermore,TFAM could be recruited by the HSP60-Lon complex in the pupal brains (Fig.4-B). When H2O2was injected into day 1 nondiapause-destined pupae to increase ROS,the stability of the HSP60-Lon complex in the brains was enhanced,and it recruited more TFAM protein,although HSP60 expression was not significantly different from the control (Fig.4-C).We further down-regulated the expression of HSP60 using RNAi in the HzAm1 cells,and found that the binding ability of Lon with TFAM was significantly decreased under oxidative stress (Fig.4-D). This indicated that HSP60 has an essential role in the recruitment of Lon and its targets in the mitochondria in diapause-destined pupal brains.

Fig.4 Reactive oxygen species (ROS) control of mitochondrial activity by enhancing HSP60 and Lon stability,increasing its ability to degrade mitochondrial transcription factor A (TFAM). A,HSP60 physically associates with Lon by Co-IP in brains. IgG was used as a negative control. B,TFAM physically combines with HSP60/Lon complex by Co-IP in brains. Brain lysates were immunoprecipitated with TFAM antibody,and then immunoblotted with anti-HSP60,Lon or TFAM antibody,respectively. C,ROS enhancement of HSP60/Lon complex binding to TFAM. Day 1 nondiapause-destined pupae were injected with 3 μL of 2 mmol L-1 H2O2 for 2 h. Brain lysates were immunoprecipitated with anti-HSP60 antibody,and then immunoblotting (IB) with the indicated antibody,respectively. WCE,whole cell extract. D,knockdown of HSP60 decreases the combination between Lon and TFAM under oxidative stress.+and -represent transfection of HSP60 dsRNA and no transfection,respectively. HzAm1 cells were transfected with 4 μg of HSP60 dsRNA for 48 h,followed by treatment with 200 μmol L-1 H2O2 for 30 min. Cell lysates were immunoprecipitated with Lon antibody,and then IB with the indicated antibody,respectively.

4.Discussion

Diapause,a long-lived “non-aging” state,is a term used for describing the slow development as a dauer inC.elegans(Hu 2007) and lifespan extension inDrosophila(Tataret al.2001). As an important regulator,ROS can promote the progress of diapause by regulating the insulin signaling pathway inH.armigera(Zhanget al.2017). However,the source of ROS in the brains and the molecular mechanism of the influence of ROS on diapause are still unclear.

In this study,we first showed that mtROS and total ROS were significantly higher in diapause-destined pupal brains than in nondiapause-destined pupal brains,which are consistent with previous report (Zhanget al.2017). These results further supported the viewpoint that mitochondria are indeed the main site of ROS generation (Ristow and Zarse 2010). In addition,through the combined analysis of the SOD and catalase activities,we speculated that the high generation of mtROS and a low antioxidant system could result in high total ROS accumulation in the diapausedestined pupal brains.

Decreased glucose utilization can increase mtROS generation inC.elegans(Schulzet al.2007). Similarly,after injection of DOG into day 1 nondiapause-destined pupae ofH.armigera,reduction of glucose utilization significantly increased mtROS in the brains,and delayed pupal development. Interestingly,the injection of NAC to eliminate ROS could rescue the delaying effect,suggesting that the mtROS play a critical role in diapausing pupae. In contrast,injection of the metabolite mixture significantly decreased mtROS levels and then channeled diapausedestined pupae into development. These results indicated that the pupae ofH.armigeracould reduce the utilization of glucose in the brain,by maintaining low levels of glucose in the hemolymph (Xuet al.2012),and thus promote mtROS generation and initiate the diapause process.

Then the little mermaid, who was very anxious to see whether she was really beautiful, was obliged to acknowledge that she had never seen a more perfect vision of beauty

Mitochondria,as the cellular energy factories,regulate many biological processes,including cellular senescence,apoptosis,and lifespan extension (Maletet al.2006;Monaghanet al.2015;Vasileiouet al.2019). Downregulation of mitochondrial activity results in low brain metabolic activity and induces diapause initiation (Denlinger 2002;Hahn and Denlinger 2011). InH.armigeradiapausedestined pupal brains,the levels of many mitochondrial proteins are significantly lower than in nondiapause-destined pupal brains (Lu and Xu 2010;Linet al.2016;Wanget al.2018;Genget al.2021). This down-regulation of mitochondrial proteins results in the suppression of mitochondrial activity. Heat shock proteins have been reported to function in regulating diapause in response to heat,cold and oxidative stress (Rinehartet al.2007;Popovicet al.2015;Zhanget al.2016). We found that the HSP60 expression levels were significantly higher in diapause-destined pupal brains than in nondiapausedestined pupae. Further experimentsinvivoandinvitrodemonstrated that HSP60 is located in mitochondria,and the expression of HSP60 was in response to mtROS in the pupal brains. These results suggest that high HSP60 expression in mitochondria is closely correlated with diapause initiation.

Although HSP60 is correlated with diapause,the mechanism still needs to be determined. One possibility is that high levels of mtROS damage mitochondrial proteins by oxidizing them,and HSP60 recognizes the damaged mitochondrial proteins and then promotes their degradation in coordination with ATP-dependent proteases (Chenget al.1989;Benderet al.2011;Fanet al.2020). In this study,we showed that HSP60 combined with Lon to form a complex in the pupal brains,and that TFAM could be recruited by the HSP60/Lon complex in the pupal brains. Furthermore,the stability of the HSP60/Lon complex was enhanced under oxidative stress,because injection of H2O2could increase the combination of HSP60 and Lon,and then recruit more TFAM. Most importantly,the ability of Lon to bind to its targets,such as TFAM,was dependent on HSP60,because silencing the expression of HSP60 decreased the binding of Lon and TFAM in the cells.

In summary,we propose a specific scenario for the regulation of mtROS/HSP60-controlled insect diapause. As a pri mary event,the direct generation of mtROS in response to decreased glucose utilization oxidatively modified the proteins in the mitochondria,such as TFAM,whereas the HSP60/Lon complex acts like a quality control valve under stress conditions,by interacting with the damaged proteins.Lon then acts as a “clean-up” protease,removing TFAM protein and decreasing the mitochondria biogenesis and activity (Linet al.2016). The low biogenesis and activity of mitochondria lead to low metabolic activity,which then leads the insect into diapause and lifespan extension.

5.Conclusion

High levels of mtROS accumulated in theH.armigeradiapause-destined pupal brain in response to decreased glucose utilization. The stability of HSP60/Lon was enhanced by ROS,increasing its capacity for removing the mitochondrial proteins,such as TFAM. This study may provide important information for further understanding of the regulatory mechanism of mtROS during insect diapause.

Acknowledgements

This study was supported by the China Postdoctoral Science Foundation (2017M622872). We are very grateful to Dr.An Shiheng (Henan Agricultural University,China),for providingHelicoverpa armigera.

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm