Guohua Zhong

Name:

Organization: South China Agricultural University

Department: College of Natural Resources and Environment

Title:

Chemicals
References

Structure-activity relationships of 3-O-β-chacotriosyl oleanic acid derivatives as entry inhibitors for highly pathogenic H5N1 influenza virus

Co-reporter:Sumei Li, Xiuhua Jia, Xintian Shen, Zhuwen Wei, Zhiyan Jiang, Yixian Liao, Yiming Guo, Xiaojun Zheng, Guohua Zhong, Gaopeng Song

Bioorganic & Medicinal Chemistry 2017 Volume 25, Issue 16(Issue 16) pp:

Publication Date(Web):15 August 2017

DOI:10.1016/j.bmc.2017.06.025

•A series of oleanane-type triterpenes were designed and synthesized.•These compounds as H5N1 entry inhibitors exhibited strong inhibitory activity.•Intensive SARs studies were conducted.•Introduction of an oxo group to position 11 at OA can enhance the SI.•Alteration of the C-3 configuration of OA can improve the selective index.Highly pathogenic H5N1 virus (H5N1) entry is a key target for the development of novel anti-influenza agents with new mechanisms of action. In our continuing efforts to identify novel potential anti-H5N1 entry inhibitors, a series of 3-O-β-chacotriosyl oleanolic acid analogs have been designed, synthesized and evaluated as H5N1 entry inhibitors based on two small molecule inhibitors 1 and 2 previously discovered by us. The anti-H5N1 entry activities were determined based on HA/HIV and VSVG/HIV entry assays. Compound 15 displayed the most promising anti-H5N1 entry activities with average IC50 values of 4.05 μM and good selective index (22.9). Detailed structure-activity relationships (SARs) studies suggested that either the introduction of an additional oxo group to position 11 at OA or alteration of the C-3 configuration of OA from 3β- to 3α-forms can significantly enhance the selective index while maintaining their antiviral activities in vitro. Molecular simulation analysis confirmed that the compounds exert their inhibitory activity through binding tightly to hemagglutinin (HA2) protein near the fusion peptide and prevent virus entry.Download high-res image (77KB)Download full-size image

Curcumin induces autophagic cell death in Spodoptera frugiperda cells

Co-reporter:Sethuraman Veeran, Benshui Shu, Gaofeng Cui, Shengjiao Fu, Guohua Zhong

Pesticide Biochemistry and Physiology 2017 Volume 139(Volume 139) pp:

Publication Date(Web):1 June 2017

DOI:10.1016/j.pestbp.2017.05.004

•Curcumin inhibited the growth of Sf9 cells.•Curcumin induced autophagic cell death in Sf9 insect cell line.•SfAtg8/LC3 level increased in a time dependent manner upon curcumin treated Sf9 cells.The increasing interest in the role of autophagy (type II cell death) in the regulation of insect toxicology has propelled study of investigating autophagic cell death pathways. Turmeric, the rhizome of the herb Curcuma longa (Mañjaḷ in Tamil, India and Jiānghuáng in Chinese) have been traditionally used for the pest control either alone or combination with other botanical pesticides. However, the mechanisms by which Curcuma longa or curcumin exerts cytotoxicity in pests are not well understood. In this study, we investigated the potency of Curcuma longa (curcumin) as a natural pesticide employing Sf9 insect line. Autophagy induction effect of curcumin on Spodoptera frugiperda (Sf9) cells was investigated using various techniques including cell proliferation assay, morphology analysis with inverted phase contrast microscope and Transmission Electron Microscope (TEM) analysis. Autophagy was evaluated using the fluorescent dye monodansylcadaverine (MDC). Cell death measurement was examined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) within the concentrations of 5–15 μg/mL. Curcumin inhibited the growth of the Sf9 cells and induced autophagic cell death in a time and dose dependent manner. Staining the cells with MDC showed the presence of autophagic vacuoles while increased in a dose and time dependent manner. At the ultrastructural level transmission electron microscopy, cells revealed massive autophagy vacuole accumulation and absence of chromatin condensation. Protein expression levels of ATG8-I and ATG8-II, well-established markers of autophagy related protein were elevated in a time dependent manner after curcumin treatment. The present study proves that curcumin induces autophagic cell death in Sf9 insect cell line and this is the first report of cytotoxic effect of curcumin in insect cells and that will be utilized as natural pesticides in future.Download high-res image (162KB)Download full-size image

Identification of multi-insecticide residues using GC-NPD and the degradation kinetics of chlorpyrifos in sweet corn and soils

Co-reporter:Peidan Wang, Muhammad Rashid, Jie Liu, Meiying Hu, Guohua Zhong

Food Chemistry 2016 Volume 212() pp:420-426

Publication Date(Web):1 December 2016

DOI:10.1016/j.foodchem.2016.05.008

•The retention time of 12 pesticides determined by GC-NPD.•The percent recoveries demonstrated that the analytical method is acceptable.•Half-life of chlorpyrifos degradation in sweet corn and soil samples were assessed.•The result were report the degradation of chlorpyrifos in sweet corn and soils.Because more than one insecticide is applied to crops to protect plants from pests, an analytical multi-residue determination method was developed using gas chromatography with a nitrogen phosphorus detector (GC-NPD). The retention time for 12 insecticides was 3.7–27.7 min. Under the selected conditions, the limits of detection (LOD) and quantification (LOQ) were below the maximum residue limits (MRLs) and in the range of 0.00315–0.05 μg mL−1 and 0.01–0.165 μg mL−1, respectively. Using GC-NPD, we investigated the dissipation dynamics and final residual levels of chlorpyrifos in sweet corn and soil and determined that the half-lives was 4–7 days, that is, that chlorpyrifos is safe to use on sweet corn with a pre-harvest interval of 16–22 days before harvest. These results provide new insights into chlorpyrifos degradation in plants and its environmental behavior.

Retraction Note to: Involvement of a Specific Chemosensory Protein from Bactrocera dorsalis in Perceiving Host Plant Volatiles

Co-reporter:Xin Yi;PeiDan Wang;Zheng Wang;Jun Cai;MeiYing Hu

Journal of Chemical Ecology 2016 Volume 42( Issue 5) pp:461

Publication Date(Web):2016 May

DOI:10.1007/s10886-016-0707-x

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

Co-reporter:Benshui Shu;Wenxiang Wang;Qingbo Hu;Jingfei Huang;Meiying Hu

Archives of Insect Biochemistry and Physiology 2015 Volume 89( Issue 3) pp:153-168

Publication Date(Web):

DOI:10.1002/arch.21233

The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.

Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its Potential for Bioremediation of Pyrethroid-Contaminated Soils

Co-reporter:Shaohua Chen, Changqing Chang, Yinyue Deng, Shuwen An, Yi Hu Dong, Jianuan Zhou, Meiying Hu, Guohua Zhong, and Lian-Hui Zhang

Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 10) pp:2147-2157

Publication Date(Web):February 20, 2014

DOI:10.1021/jf404908j

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L–1 fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h–1, 9.0 mg L–1, and 694.8 mg L–1, respectively. Analysis of the degradation products by gas chromatography–mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.

MOLECULAR CLONING AND CHARACTERIZATION OF A SID-1-LIKE GENE IN Plutella xylostella

Co-reporter:Huidong Wang;Liang Gong;Jiangwei Qi;Meiying Hu;Liang Gong

Archives of Insect Biochemistry and Physiology 2014 Volume 87( Issue 3) pp:164-176

Publication Date(Web):

DOI:10.1002/arch.21189

RNA interference (RNAi) signal can spread from the point where the double-stranded RNA (dsRNA) was initially applied to other cells or tissues. SID-related genes in Caenorhabditis elegans help in the spreading of this signal. However, the mechanisms of systemic RNAi are still not unveiled in insects. In this study, we cloned a full-length cDNA of sid-1-like gene, Pxylsid-1, from Plutella xylostella that contains 1,047 bp opening reading frame encoding a putative protein of 348 amino acids. This transcript is very much similar to the sil-1 in Bombyx mori (68.8%). The higher expression levels of Pxylsid-1 were found at the adult and fourth-instar stages compared to the second-instar stage with 21.48- and 10.36-fold increase, respectively. Its expression levels in different tissues were confirmed with the highest expression in the hemolymph, which showed 21.09-fold increase than the midgut; however it was lower in other tissues. The result of RNAi by feeding bacterially expressed dsRNA targeting Pxylace-1, which showed that the mRNA level of Pxylace-1 decreased by 34.52 and 64.04% after 36- and 72-h treatment, respectively. However, the mRNA level of Pxylsid-1 was not significantly induced when the Pxylace-1 was downregulated. Furthermore, we found that downregulation of Pxylsid-1 did not affect the RNAi effect of Pxylace-1. Hence, the Pxylsid-1 may not be involved in absorption of dsRNA from the midgut fluid. A further study is needed to uncover the function of Pxylsid-1.

CONTACTING IS ESSENTIAL FOR OVIPOSITION DETERRENCE OF RHODOJAPONIN-III IN SPODOPTERA LITURA

Co-reporter:Xin Yi;Jinxiang Liu;Peidan Wang;Meiying Hu

Archives of Insect Biochemistry and Physiology 2014 Volume 86( Issue 2) pp:122-136

Publication Date(Web):

DOI:10.1002/arch.21170

In Lepidoptera, choosing the right site for egg laying is particularly important, because the small larvae cannot forage for alternate host plants easily. Some secondary compounds of plants have the ability to deter oviposition behaviors of insects. Rhodojaponin-III, a botanical compound, has been reported to have intense deterring-oviposition activity against many insects, which have important implications for agricultural pest management. This study provided evidence for elucidating the perception mechanism underlying Rhodojaponin-III as oviposition deterrent. In this study, the antennas of moths could not elicit notable electroantennogram responses to Rhodojaponin-III, which suggested the Rhodojaponin-III could not exert effects like those volatile compounds. The results of physiological experiments confirmed the Rhodojaponin-III could produce the oviposition deterrence effect against moths without depending on antennas, while the physical contact was essential for perceiving the compound, which suggested that the sensilla on tarsus and ovipositor could be chemoreceptor for Rhodojaponin-III. Therefore, these sensilla were investigated by scanning electron microscopy to explore their potential functions in detecting Rhodojaponin-III. This study highlighted the contacting mechanism in deterring oviposition behaviors of moths by Rhodojaponin-III and provided new insight for development of contact-based pest management.

RETRACTED ARTICLE: Involvement of a Specific Chemosensory Protein from Bactrocera dorsalis in Perceiving Host Plant Volatiles

Co-reporter:Xin Yi;PeiDan Wang;Zheng Wang;Jun Cai;MeiYing Hu

Journal of Chemical Ecology 2014 Volume 40( Issue 3) pp:267-275

Publication Date(Web):2014 March

DOI:10.1007/s10886-014-0406-4

Insects have evolved many physiological and behavioral adaptations to recognize external complex chemicals. Olfaction plays an important role in perceiving volatile chemicals, utilizing them to locate host sites, conspecifics, and enemies. Chemosensory proteins (CSPs) are present in high concentrations within the sensory sensilla of insects and are endowed with a heterogeneous range of functions. However, direct evidence for the involvement of CSPs in olfactory function is still lacking. In this study, a fluorescence-based ligand binding assay using Bdor-CSP2 illustrated its ability to bind the majority of the selected ligands of different shapes and chemical structures that are ecologically significant, host plant volatiles of Bactrocera dorsalis. RNAi-mediated silencing coupled with electrophysiological tests showed lower electrophysiological responses to (3Z)-hex-3-en-1-ol, trans-2-hexenal, 6-methylhept-5-en-2-one, and 3-methylbutyl acetate in dsBdor CSP2 treated flies compared with the untreated controls. The reduced expression of Bdor-CSP2 by RNA interference was confirmed by semi-quantitative PCR, real-time quantitative PCR and Western blot, which suggested the RNAi-treatment was responsible for the observed reduction of antennal responses in EAG recordings. These data suggest that the expression of Bdor-CSP2 is necessary for the recognition of antennal responses to some plant host volatiles by B. dorsalis.

Resistance to fenpropathrin, chlorpyriphos and abamectin in different populations of Amblyseius longispinosus (Acari: Phytoseiidae) from vegetable crops in South China

Co-reporter:Hai-ming Zhao, Xin Yi, Yue-ye Deng, Mei-ying Hu, Guo-hua Zhong, Pei-dan Wang

Biological Control (October 2013) Volume 67(Issue 1) pp:61-65

Publication Date(Web):October 2013

DOI:10.1016/j.biocontrol.2013.07.007

BdorOrco is important for oviposition-deterring behavior induced by both the volatile and non-volatile repellents in Bactrocera dorsalis (Diptera: Tephritidae)

Co-reporter:Xin Yi, Haiming Zhao, Peidan Wang, Meiying Hu, Guohua Zhong

Journal of Insect Physiology (June 2014) Volume 65() pp:51-56

Publication Date(Web):1 June 2014

DOI:10.1016/j.jinsphys.2014.05.007

•Citronella oil and Rhodojaponin-III have intense oviposition-deterring activity.•Bdor⧹Orco and Bdor⧹OBP were involved in the detection of volatile oviposition repellents.•Silencing the expression of Bdor⧹Orco resulted in disoriented oviposition behaviors induced by two repellents.Several studies have shown that the selections of gravid females to potential oviposition sites from a distance were mediated by volatile signals, however, the means by which the sensory cues from non-volatile chemicals affected the insect behavior were still a controversial subject. Chemosensory in insect is a complex process, which is mediated by multigene families of chemoreceptors, including olfactory receptors, olfactory co-receptors, and odorant-binding proteins. To elucidate the chemoreception mechanism of volatile and non-volatile chemicals, the roles of Orco and OBP in oviposition-deterrent activities induced by citronellal and Rhodojaponin-III were investigated. Our results suggested that RNAi-mediated expression inhibition was successfully achieved by feeding dsRNA in Bactrocera dorsalis. High levels of Bdor⧹Orco expression were essential for recognizing two chemicals of different physical properties, whereas the expression of Bdor⧹OBP was only imperative in perception of volatile chemical. The results suggested that volatile and non-volatile chemicals may evoke distinct molecular basis for chemosensory in the flies, while Orco was essential in the perception of both chemicals. The study highlighted that the central role of Orco in chemical recognition, which enabled it to be the universally applied target of designing new botanical pesticide.Graphical abstractDownload full-size image

Preliminary studies on induction of apoptosis by abamectin in Spodoptera frugiperda (Sf9) cell line

Co-reporter:Jing-Fei Huang, Meng Tian, Chao-Jun Lv, Hai-Yi Li, Rizwan-ul-Haq Muhammad, Guo-Hua Zhong

Pesticide Biochemistry and Physiology (July 2011) Volume 100(Issue 3) pp:256-263

Publication Date(Web):1 July 2011

DOI:10.1016/j.pestbp.2011.04.010

Avermectins are a series 16-membered macrocyclic lactone derivatives derived from the actinomycete Streptomyces avermectinius, which has been applied worldwide in veterinary, human medicine and agriculture. Abamectin is a mixture of avermectins containing ⩾80% avermectin B1a and ⩽20% avermectin B1b. In this paper, the apoptotic induction effect of abamectin on Spodoptera frugiperda (Sf9) cells was detected by using various techniques including cell proliferation assay, flow cytometry, morphology analysis with inverted phase contrast microscope, scanning electron microscope, transmission electron microscopy, enzyme activity assay and Western blot. The results revealed that within the concentrations of 5–15 μg/mL, abamectin inhibited the growth of the Sf9 cells and induced apoptotic cell death in a time- and dose-dependent manner. The proliferation inhibition rates of Sf9 cells were 6.45 ± 0.47%, 11.51 ± 0.20% and 12.97 ± 0.46% after 12 h of treatment with avermectin at the concentrations of 5, 10 and 15 μg/mL, which increased to 30.74 ± 0.57%, 36.14 ± 0.25% and 39.27 ± 0.63% after 72 h of the same treatments, respectively. The maximum apoptosis rate of each concentration was 24.13 ± 0.43%, 24.69 ± 0.38% and 27.285 ± 0.84%, respectively. Treated cells showed typical apoptosis morphological changes including cell swelling, chromatin condensation, apoptotic bodies, swollen mitochondria and high content of phagosome. Both mitochondrial events including mitochondrial membrane potential (Δψm) loss as well as cytochrome-c release into cytosol and significant activation of caspase-3 occurred during apoptosis. These data confirmed the apoptosis inducing effects of abamectin on S. frugiperda (Sf9) cell line, and provide preliminary studies on its mechanism.Graphical abstractDownload full-size imageHighlights► Loss of mitochondrial membrane potential. ► Cytochrome-c release from mitochondria to cytosol. ► Activation of caspase-3.

Antiproliferative effect of azadirachtin A on Spodoptera litura Sl-1 cell line through cell cycle arrest and apoptosis induced by up-regulation of p53

Co-reporter:Jing-Fei Huang, Ke-Juan Shui, Hai-Yi Li, Mei-Ying Hu, Guo-Hua Zhong

Pesticide Biochemistry and Physiology (January 2011) Volume 99(Issue 1) pp:16-24

Publication Date(Web):1 January 2011

DOI:10.1016/j.pestbp.2010.08.002

In order to illustrate the mechanism of cell cycle arrest and apoptosis induced by botanical insecticide, the effects of cell proliferation inhibition and apoptosis induction by azadirachtin A were investigated in Spodoptera litura cell line Sl-1 in vitro. The cell proliferation, cell cycle division, mitotic and apoptotic figure of nucleus, and the expression of related proteins were detected by a variety of methods, including MTT assay, flow cytometry, fluorescence microscope observation, and Western blot technique. Sl-1 cells grew in a dose-dependent inhibition manner and the proliferation inhibition rates were 18.95%, 33.45% and 41.00%, after 48 h of treatment with azadirachtin A at the concentrations of 0.1, 0.5 and 1.0 μg/mL, respectively. Sl-1 cell cycle was arrested in G2/M phase with lower expression of cyclin B1. The apoptosis rates were found to be 19.71%, 23.40% and 24.91% after 48 h of treatment with 0.1, 0.5, and 1.0 μg/mL of azadirachtin A, which increased with the activities of caspase-3. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. These findings suggest that p53 protein was involved in cell cycle arrest, apoptosis induction and cell proliferation inhibition when the insect cultured cells were treated with azadirachtin A.Graphical abstractDownload full-size imageResearch highlights► Inhibitory effect of azadirachtin A on the proliferation of Sl-1 cells. ► Azadirachtin A induced G2/M arrest and apoptosis in the Sl-1 cells. ► Azadirachtin A down-regulated expression of cyclin B1. ► Azadirachtin A up-regulated expression of p53 and activated caspase-3.

Combination of hot water, Bacillus amyloliquefaciens HF-01 and sodium bicarbonate treatments to control postharvest decay of mandarin fruit

Co-reporter:Peng Hong, Weining Hao, Jianjun Luo, Shaohua Chen, Meiying Hu, Guohua Zhong

Postharvest Biology and Technology (February 2014) Volume 88() pp:96-102

Publication Date(Web):1 February 2014

DOI:10.1016/j.postharvbio.2013.10.004

•2% sodium bicarbonate (SBC) had no clear negative effect on Bacillus amyloliquefaciens strain HF-01 survival.•The combination of strain HF-01, SBC and hot water significantly reduced postharvest decay on citrus.•This combined treatment also reduced natural decay development of mandarin fruit.•The combined treatment did not impair the quality parameters of fruit.An antagonistic isolate Bacillus amyloliquefaciens HF-01, sodium bicarbonate (SBC) and hot water treatment (HW) were investigated individually and in combination against green and blue mold and sour rot caused by Penicillium digitatum, P. italicum and Geotrichum citri-aurantii respectively, in mandarin fruit. Populations of antagonists were stable in the presence of 1% or 2% SBC treatment, and spore germination of pathogens in potato dextrose broth was greatly controlled by the hot water treatment of 45 °C for 2 min. Individual application of sodium bicarbonate at low rates and hot water treatment, although reducing disease incidence after 8 weeks or 4 weeks of storage at 6 °C or 25 °C respectively, was not as effective as the fungicide treatment. The treatment comprising B. amyloliquefaciens combined with 2% SBC or/and HW (45 °C for 2 min) was as effective as the fungicide treatment and reduced decay to less than 80% compared to the control. B. amyloliquefaciens HF-01 alone or in combination with 2% SBC or/and HW significantly reduced postharvest decay without impairing fruit quality after storage at 25 °C for 4 weeks or at 6 °C for 8 weeks. These results suggest that the combination of B. amyloliquefaciens HF-01, SBC and HW could be a promising method for the control of postharvest decay on citrus while maintaining fruit quality after harvest.

Iron–sulfur protein in mitochondrial complexes of Spodoptera litura as potential site for ROS generation

Co-reporter:Liangde Li, Xiaolin Dong, Benshui Shu, Zheng Wang, Qiongbo Hu, Guohua Zhong

Journal of Insect Physiology (December 2014) Volume 71() pp:21-29

Publication Date(Web):1 December 2014

DOI:10.1016/j.jinsphys.2014.08.009

•The ISCU2 and ISCU8 genes were cloned from Spodoptera litura.•The developmental expression patterns of ISCUs in the S. litura were examined.•RNA interference of ISCUs could result in declining of the ROS in Sf9 cells.•Rotenone and azadirachtin treatment could increase Sf9 ISCUs and ROS levels.•SOD and POD were increased by rotenone and azadirachtin and declined by ISCU-siRNA.Mitochondrial complex I is the main source of reactive oxygen species (ROS) production, but the exact site of superoxide generation or their relative contribution is not clear. This study aims to determine the function of iron–sulfur clusters (ISCU) in the initiation of ROS generation. ISCU2 and ISCU8 were cloned from Spodoptera litura which shared the conserved amino acid sequence with other insects. The expressions of the two genes were ubiquitous throughout the whole development stages and tissues. Knockdown of ISCU2 and ISCU8 resulted in the decline of the ROS, whereas rotenone and azadirachtin treatment up-regulated ROS levels by increasing mRNA expression. Furthermore, antioxidant enzyme activity of SOD and POD were up-regulated by rotenone and azadirachtin treatment and then declined after ISCU was silenced. Our results suggest the possibility that the molecules of ISCU2 and ISCU8 in complex I may serve as potential sites in the initiation of ROS generation.Download full-size image