Loss of AMPK activity induces organelle dysfunction and oxidative stress during oocyte aging.

BACKGROUND: Oocyte quality is critical for the mammalian reproduction due to its necessity on fertilization and early development. During aging, the declined oocytes showing with organelle dysfunction and oxidative stress lead to infertility. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which is important for energy homeostasis for metabolism. Little is known about the potential relationship between AMPK with oocyte aging. RESULTS: In present study we reported that AMPK was related with low quality of oocytes under post ovulatory aging and the potential mechanism. We showed the altered AMPK level during aging and inhibition of AMPK activity induced mouse oocyte maturation defect. Further analysis indicated that similar with its upstream regulator PKD1, AMPK could reduce ROS level to avoid oxidative stress in oocytes, and this might be due to its regulation on mitochondria function, since loss of AMPK activity induced abnormal distribution, reduced ATP production and mtDNA copy number of mitochondria. Besides, we also found that the ER and Golgi apparatus distribution was aberrant after AMPK inhibition, and enhanced lysosome function was also observed. CONCLUSIONS: Taken together, these data indicated that AMPK is important for the organelle function to reduce oxidative stress during oocyte meiotic maturation.

Microcapsules of mesoporous silica and cyclodextrin modified loaded with nonanal and decanal for effective control of Sitotroga cerealella in grain storage environments.

BACKGROUND: The Angoumois grain moth, Sitotroga cerealella, is a destructive pest of maize, wheat, and rice, causing economic losses and threatening food security. This study aimed to develop and characterize microcapsules of mesoporous silica nanospheres (MSN) and cyclodextrin-modified mesoporous silica nanospheres (CDMSN) containing two aldehydes, nonanal and decanal, found in plant essential oils, to assess their attractiveness to S. cerealella populations. RESULTS: Microcapsules with 2:1 ratio of nonanal and decanal exhibited an average encapsulation efficiency of 39.82% for MSN loaded with nonanal and decanal (MSN-ND) and 46.10% for CDMSN loaded with nonanal and decanal (CDMSN-ND). They have an elliptical shape with particle sizes of 115 nm for MSN and 175 nm for CDMSN. Gas chromatography-mass spectrometry analysis revealed in vitro release of nonanal in MSN at 96.24% and decanal at 96.42% by the 36th day. CDMSN showed releases of 93.83% for nonanal and 93.74% for decanal by the 50th day. CDMSN-ND attracted adult S. cerealella for 43 days, while MSN-ND remained effective for 29 days. In mass trapping assays in simulated grain warehouse, both MSN-ND and CDMSN-ND trapped over 50% of the adult population within 7 days, significantly reducing grain infestation rates below 10% by inhibiting F1 adult emergence. At temperatures ranging from 20 °C to 35 °C, both microcapsules exhibited significant and effective attraction rates for S. cerealella. Stored wheat seeds treated with CDMSN and CDMSN-ND over 1 year showed no significant differences in key germination parameters. CONCLUSION: Microencapsulated nonanal and decanal offer a promising, sustainable approach for controlling S. cerealella infestation in stored grains, contributing to global food security. © 2024 Society of Chemical Industry.

Discussion: Harnessing microbiome-mediated adaptations in insect pollinators to mitigate climate change impact on crop pollination.

Insect pollinators, vital for agriculture and biodiversity, face escalating threats from climate change. We argue and explore the pivotal role of the microbiomes in shaping adaptations of insect pollinator resilience amid climate-induced challenges (climate change and habitat alteration). Examining diverse taxonomic groups, we unravel the interplay between insect physiology, microbiomes, and adaptive mechanisms. Climate-driven alterations in microbiomes impact insect health, behavior, and plant interactions, posing significant effects on agricultural ecosystems. We propose harnessing microbiome-mediated adaptations as a strategic approach to mitigate climate change impacts on crop pollination. Insights into insect-pollinator microbiomes offer transformative avenues for sustainable agriculture, including probiotic interventions (use of EM PROBIOTIC) and microbiome engineering (such as engineering gut bacteria) to induce immune responses and enhanced pollination services. Integrating microbiome insights into conservation practices elucidates strategies for preserving pollinator habitats, optimizing agricultural landscapes, and developing policies to safeguard pollinator health in the face of environmental changes. Finally, we stress interdisciplinary collaboration and the urgency of understanding pollinator microbiome dynamics under climate change in future research.

Diallyl Trisulfide Causes Male Infertility with Oligoasthenoteratospermia in Sitotroga cerealella through the Ubiquitin-Proteasome Pathway.

Essential oils extracted from plant sources along with their biologically active components may have negative effects on insects. Diallyl trisulfide (DAT) is an active component of garlic essential oil, and it exhibits multi-targeted activity against many organisms. Previously we reported that DAT induces male infertility and leads to apyrene and eupyrene sperm dysfunction in Sitotroga cerealella. In this study, we conducted an analysis of testis-specific RNA-Seq data and identified 449 downregulated genes and 60 upregulated genes in the DAT group compared to the control group. The downregulated genes were significantly enriched in the ubiquitin-proteasome pathway. Furthermore, DAT caused a significant reduction in mRNA expression of proteasome regulatory subunit particles required for ATP-dependent degradation of ubiquitinated proteins as well as decreased the expression profile of proteasome core particles, including ß1, ß2, and ß5. Sperm physiological analysis showed that DAT decreased the chymotrypsin-like activity of the 20S proteasome and formed aggresomes in spermatozoa. Overall, our findings suggest that DAT impairs the testis proteasome, ultimately causing male infertility characterized by oligoasthenoteratospermia due to disruption in sperm proteasome assembly in S. cerealella.

Uncovering the ceRNA network and DNA methylation associated with gene expression in nasopharyngeal carcinoma.

OBJECTIVE: This study aimed to uncover abnormally expressed genes regulated by competitive endogenous RNA (ceRNA) and DNA methylation nasopharyngeal carcinoma and to validate the role of lncRNAs in the ceRNA network on nasopharyngeal carcinoma progression. METHODS: Based on the GSE64634 (mRNA), GSE32960 (miRNA), GSE95166 (lncRNA), and GSE126683 (lncRNA) datasets, we screened differentially expressed mRNAs, miRNAs and lncRNAs in nasopharyngeal carcinoma. A ceRNA network was subsequently constructed. Differentially methylated genes were screened using the GSE62336 dataset. The abnormally expressed genes regulated by both the ceRNA network and DNA methylation were identified. In the ceRNA network, the expression of RP11-545G3.1 lncRNA was validated in nasopharyngeal carcinoma tissues and cells by RT-qPCR. After a knockdown of RP11-545G3.1, the viability, migration, and invasion of CNE-2 and NP69 cells was assessed by CCK-8, wound healing and Transwell assays. RESULTS: This study identified abnormally expressed mRNAs, miRNAs and lncRNAs in nasopharyngeal carcinoma tissues. A ceRNA network was constructed, which contained three lncRNAs, 15 miRNAs and 129 mRNAs. Among the nodes in the PPI network based on the mRNAs in the ceRNA network, HMGA1 was assessed in relation to the overall and disease-free survival of nasopharyngeal carcinoma. We screened two up-regulated genes regulated by the ceRNA network and hypomethylation and 26 down-regulated genes regulated by the ceRNA network and hypermethylation. RP11-545G3.1 was highly expressed in the nasopharyngeal carcinoma tissues and cells. Moreover, the knockdown of RP11-545G3.1 reduced the viability, migration, and invasion of CNE-2 and NP69 cells. CONCLUSION: Our findings uncovered the epigenetic regulation in nasopharyngeal carcinoma and identified the implications of RP11-545G3.1 on the progression of nasopharyngeal carcinoma.

Microplastics and Nanoplastics Effects on Plant-Pollinator Interaction and Pollination Biology.

Microplastics and nanoplastics (MNPs) contamination is an emerging environmental and public health concern, and these particles have been reported both in aquatic and terrestrial ecosystems. Recent studies have expanded our understanding of the adverse effects of MNPs pollution on human, terrestrial, and aquatic animals, insects, and plants. In this perspective, we describe the adverse effects of MNPs particles on pollinator and plant health and discuss the mechanisms by which MNPs disrupt the pollination process. We discuss the evidence and integrate transcriptome studies to investigate the negative effects of MNPs on the molecular biology of pollination, which may cause delay or inhibit the pollination services. We conclude by addressing challenges to plant-pollinator health from MNPs pollution and argue that such harmful effects disrupt the communication between plant and pollinator for a successful pollination process.

Diallyl Trisulfide, a Biologically Active Component of Garlic Essential Oil, Decreases Male Fertility in Sitotroga cerealella by Impairing Dimorphic Spermatogenesis, Sperm Motility and Lipid Homeostasis.

Diallyl trisulfide (DAT) is a biologically active component of garlic essential oil and exhibits multi-targeted activity against many organisms. The current study tested the capacity of DAT to decrease the male fertility of Sitotroga cerealella. The effects on testis morphology, sperm number, motility, and lipid homeostasis were observed in adult males fumigated with DAT at a dose of 0.01 µL/L in air. The results indicated that the DAT significantly decreased the dimorphic sperm number. Meanwhile, the ultrastructural analysis of the sperm showed that the DAT caused malformed and aberrant structures of mitochondrial derivatives of dimorphic sperm. Additionally, the lipid homeostasis and ATP contents in the male adults were significantly decreased after treatment. Moreover, the total sperm motility was reduced, while the wave-propagation velocity, amplitude, frequency, and wavelength were significantly decreased compared with the controls. Overall, this study reported, for the first time, that DAT impairs energy metabolism, inhibits dimorphic spermatogenesis, and decreases sperm motility, while these abnormalities in sperm lead to adult-male infertility.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones.

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 µL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

[Correlation of single nucleotide polymorphisms of the osteopontin gene with astheozoospermia].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphisms (SNP) rs1126772, rs117291487, rs11730582, rs142608941 and rs6813526 of the osteopontin (OPN) gene with the risk of asthenozoospermia (AZS). METHODS: We included 135 AZS patients in the AZS group and another 239 fertile men as normal controls. Using the SNaPshot technique, we genotyped the rs1126772, rs117291487, rs11730582, rs142608941 and rs6813526 polymorphisms of the OPN gene in all the subjects and analyzed the correlation of the five SNPs with AZS. RESULTS: The GA genotype and A allele of the OPN gene rs1126772 were found to be correlated with the risk of AZS (GA vs AA: OR = 0.55, 95% CI: 0.35－0.86, P = 0.009; A vs G: OR = 0.64, 95% CI: 0.46－0.89, P = 0.007), and so was the CT genotype and T allele at the RS11730582 locus (CT vs TT: OR = 0.526, 95% CI: 0.34－0.82, P = 0.009; T vs C: OR = 0.60, 95% CI: 0.44－0.83, P = 0.002). Haplotype analysis showed that the AATCT haplotype decreased the risk of AZS (AATCT: OR = 0.61, 95% CI: 0.42－0.88, P = 0.008) . CONCLUSIONS: The polymorphisms of the OPN gene RS1126772 and RS11730582 may reduce the risk of AZS.

Silencing the triacylglycerol lipase (TGL) gene decreases the number of apyrene sperm and inhibits oviposition in Sitotroga cerealella.

Triacylglycerol lipase (TGL) is an essential lipid metabolism enzyme that also plays a critical role in energy metabolism; however, how it regulates other life processes is unknown. To investigate the functional role of TGL in moth reproduction, males Sitotroga cerealella were used as a model. The TGL gene was cloned and analysed. The results showed that the open reading frame of TGL was 1968 bp long and contained three conserved regions. TGL gene expression was higher in the larval and early adult stages than in the pupal stage, with the highest levels observed in the fat body, testis and accessory glands during the early adult stage. Moreover, after TGL in male adults was silenced through RNAi, the protein content in male accessory glands remained unchanged, and the spermatophore transferred into females mated with TGL-silenced males became small and empty; meanwhile, the number of apyrene sperm in the spermatophore was significantly reduced due to the reduction of apyrene sperm in males, which eventually led to the significant reduction of egg-laying amount. All of the findings suggest that TGL regulates the amount of sperm in male moths as well as the morphology and quality of spermatophores transferred to females after mating with treated males, implying that TGL is critical for Sitotroga cerealella's reproductive process.

Normal cerebrospinal fluid protein and associated clinical characteristics in children with tuberculous meningitis.

BACKGROUND: Although abnormal cerebrospinal fluid (CSF) protein can be used to predict the outcome of tuberculous meningitis (TBM) and diagnose TBM, normal CSF protein remains a concern in patients with TBM. This retrospective study aimed to assess the clinical characteristics associated with normal CSF protein, to resolve the dilemma of CSF protein in the management of childhood TBM. METHODS: Between January 2006 and December 2019, consecutive child patients (≤15 years old, a diagnosis of TBM, and tested for CSF protein) were included for analysis. CSF protein was tested on a chemistry analyzer using the pyrogallol red-molybdate method. Abnormal CSF protein was defined as >450 mg/L. Patient characteristics were collected from the electronic medical records. Then, characteristics associated with normal CSF protein were estimated in the study, using univariate and multivariate logistic regression analysis. RESULTS: A total of 125 children who met the criteria were enrolled during the study period. Twenty-nine patients had a normal CSF protein and 96 had an abnormal CSF protein. Multivariate analysis (Hosmer-Lemeshow goodness-of-fit test: χ2=2.486, df = 8, p = .962) revealed that vomiting (age- and sex-adjusted OR = 0.253, 95% CI: 0.091, 0.701; p = .008) and serum glucose (>5.08 mmol/L; age- and sex-adjusted OR = 0.119, 95% CI: 0.032, 0.443; p = .002) were associated with the normal CSF protein in childhood TBM. CONCLUSION: In suspected childhood TBM, patients without vomiting or having low serum glucose are easy to present with normal CSF protein. Hence, when interpreting the level of CSF protein in children with such characteristics, a careful clinical assessment is required.KEY MESSAGESIn suspected childhood tuberculous meningitis, patients without vomiting or having low serum glucose are easy to present with normal CSF protein. Hence, when interpreting the level of CSF protein in children with such characteristics, a careful clinical assessment is required.

LncRNA HOTAIR regulates the expression of E-cadherin to affect nasopharyngeal carcinoma progression by recruiting histone methylase EZH2 to mediate H3K27 trimethylation.

BACKGROUND/AIM: There has been increasing evidence for the function of long non-coding RNA (lncRNA) in nasopharyngeal carcinoma (NPC). We aim to delve into the position of lncRNA HOX antisense intergenic RNA (HOTAIR), together with enhancer of zeste homolog 2 (EZH2), E-cadherin and trimethylation of lysine 27 on histone H3 (H3K27me3) in NPC. METHODS: HOTAIR, EZH2, and E-cadherin expression in NPC tissues and cells were tested. NPC cell biological functions were examined through gain-of and loss-of function assays. The mechanism of lncRNA HOTAIR/E-cadherin/EZH2/H3K27 axis in NPC was decoded. RESULTS: LncRNA HOTAIR and EZH2 were highly expressed in NPC, and E-cadherin was lowly expressed. Down-regulation of HOTAIR or EZH2 inhibited NPC cell progression and tumor growth. HOTAIR recruited histone methylase EZH2 to mediate trimethylation of H3K27 and regulated E-cadherin expression. CONCLUSION: HOTAIR inhibits E-cadherin by stimulating the trimethylation of H3K27 to promote NPC cell progression through recruiting histone methylase EZH2.

FASCIN regulates actin assembly for spindle movement and polar body extrusion in mouse oocyte meiosis.

During mouse oocyte meiotic maturation, actin filaments play multiple roles in meiosis such as spindle migration and cytokinesis. FASCIN is shown to be an actin-binding and bundling protein, making actin filaments tightly packed and parallel-aligned, and FASCIN is involved in several cellular processes like adhesion and migration. FASCIN is also a potential prognostic biomarker and therapeutic target for the treatment of metastatic disease. However, little is known about the functions of FASCIN in oocyte meiosis. In the present study, we knocked down the expression of FASCIN, and our results showed that FASCIN was essential for oocyte maturation. FASCIN was all expressed in the different stages of oocyte meiosis, and it mainly localized at the cortex of oocytes from the GV stage to the MII stage and showed a similar localization pattern with actin and DAAM1. Depletion of FASCIN affected the extrusion of the first polar body, and we also observed that some oocytes extruded from the large polar bodies. This might have resulted from the defects of actin assembly, which further affected the meiotic spindle positioning. In addition, we showed that inhibition of PKC activity decreased FASCIN expression, indicating that FASCIN might be regulated by PKC. Taken together, our results provided evidence for the important role of FASCIN on actin filaments for spindle migration and polar body extrusion in mouse oocyte meiosis.

Effects of diallyl trisulfide, an active substance from garlic essential oil, on structural chemistry of chitin in Sitotroga cerealella (Lepidoptera: Gelechiidae).

The environmental pollution, evolution of resistance, and risks to human and aquatic animal health associated with pesticide application have attracted much attention globally. Herein, we tested the capacity of diallyl trisulfide (DAT) from garlic essential oil to control the destructive stored-product pest, Sitotroga cerealella. The effects of DAT on the total content of cuticular chitin and structure of adults S. cerealella were evaluated. This study was the first to investigate changes in chitin structure in adults due to exposure to DAT through Fourier-transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, and differential scanning calorimetry. The results of these analyses revealed that the cuticular chitin content of pests decreased after DAT treatment. DAT treatment also reduced thermal stability and crystallinity of chitin. These findings indicate that DAT is a potent biopesticide that is active against the moth, and establishes the basis for its use as an IPM and alternative to chitin synthesis inhibitors.

Treatment delay in childhood pleural tuberculosis and associated factors.

BACKGROUND: Delay in diagnosis and treatment worsens the disease and clinical outcomes, which further enhances the transmission of tuberculosis (TB) in the community. Therefore, this study aims to assess treatment delay and its associated factors among childhood pleural TB patients in China. METHODS: Between January 2006 and December 2019, consecutive patients aged ≤15 years with definite or possible pleural TB were included for analysis. Treatment delay duration was defined as the time interval from the onset of symptoms to treatment initiation and was stratified into two categories: < 30 days, ≥30 days (median delay day is 30 days). The electronic medical records of children were reviewed to obtain demographic characteristics, clinical characteristics, laboratory examinations, and radiographic findings. Univariate and multivariate logistic regressions were used to explore the factors associated with treatment delay in patients. RESULTS: A total of 154 children with pleural TB were included, with a mean age of 12.4 ± 3.3 years. The median treatment delay was 30 days (interquartile range, 10-60 days) and 51.3% (n = 79) of patients underwent a treatment delay. Multivariate analysis revealed that heart rate (≤92 beats/min, age-adjusted OR = 2.503, 95% CI: 1.215, 5.155) and coefficient of variation of red cell distribution width (RDW-CV, ≥12.9%, age-adjusted OR = 4.705, 95% CI: 2.048, 10.811) were significant risk factors for treatment delays in childhood pleural TB. CONCLUSION: Our findings suggested that a significant treatment delay occurs among children with pleural TB in China. Patients with a low heart rate or a high RDW-CV experienced delays in the initiation of anti-TB therapy. Therefore, well awareness of the associations between clinical characteristics and treatment delay may improve the management of children with pleural TB and enable us to develop preventive strategies to reduce the treatment delay.

Effects of Lycium barbarum Polysaccharide on Endoplasmic Reticulum Stress and Oxidative Stress in Obese Mice.

BACKGROUND: The incidence of obesity-associated decline in male fertility has increased over the years. Lycium barbarum polysaccharide (LBP), a natural plant polysaccharide extracted from the Chinese herb L. barbarum has shown promising therapeutic effects in overcoming the same. AIM: This study aimed to investigate the protective effect of LBP on the testes of obese mice. METHODS: Following administration of LBP to high-fat diet-induced obese mice for 35 days, serum, sperm, and testis samples were obtained for subsequent experiments. Biochemical analysis and sex hormone content determination were performed to observe changes in glycolipid metabolism and testosterone levels, respectively, in the blood. Hematoxylin and eosin staining were carried out to assess the pathological changes in the testicular tissue. Oxidative stress levels were detected using enzyme-linked immunosorbent assay and expression levels of endoplasmic reticulum stress markers were determined using western blot in the testicular tissue. RESULTS: Our results suggested that LBP reduced glucose levels and insulin resistance, increased testosterone levels and insulin sensitivity, and decreased testicular oxidative stress and pathological damage in obese mice. In addition, LBP down-regulated the expression of p-eIF2α, GRP78, and CHOP in the testicular tissues of obese mice. CONCLUSION: Our results show that LBP is a potential novel drug for preventing male infertility caused by obesity.

Effects of Diallyl Trisulfide, an Active Substance from Garlic Essential Oil, on Energy Metabolism in Male Moth Sitotroga cerealella (Olivier).

This study investigated the effects of diallyl trisulfide (DAT), an active substance from garlic essential oil, on the metabolism of the main energy substances of pre- and postmating males of Sitotroga cerealella. Males at 12 h postemergence were fumigated with DAT at a concentration (LC10 = 0.010 µL/L) in a glass jar for 7 h. The main energy metabolites from pre- and postmating males were determined, including protein, triglyceride, glycogen, total soluble sugar, trehalose, and trehalase. The contents of total protein and total soluble sugar and the trehalase activity of premating males were significantly increased following DAT treatment, whereas the contents of protein from the accessory gland, triglyceride, glycogen, and trehalose were significantly decreased after treatment. Additionally, after mating, the total protein and soluble sugar contents were significantly increased and the glycogen content was significantly decreased in the treatment group relative to the levels in controls, but there was no significant difference observed in triglyceride, accessory gland proteins, trehalose content, or trehalase activity between the treatment and control groups. Furthermore, the changes in the main energy substances between pre- and postmating in males after the DAT treatment (∆DAT) were smaller than those in the control group (∆CK). This result indicated that DAT can accelerate the rate of metabolism in males at LC10, leading to the accumulation of greater levels of total soluble sugar to support life activities and to the increased synthesis of proteins to resist an adverse environment.

Time-dependent stress evidence in dynamic allocation of physiological metabolism of Nilaparvata lugens in response to elevated CO2.

To assess the time-dependent stress evidence in dynamic allocation of physiological metabolism of Nilaparvata lugens nymphs in response to elevated CO2, we measured the time-dependent allocation of nutrient compositions and physiological metabolism in the bodies of N. lugens at 1h, 4h and 12h under elevated CO2. Elevated CO2 significantly increased the contents of nutrient compositions (protein, glucose and total amino acids) and catalase (CAT) enzyme activity in the body of N. lugens at 12h relative to 1h and 4h (P < 0.05). Significantly higher genes expression levels of acetylcholinesterase (AChE), heat shock protein (HSP70) and vitellogenin gene (vg) were observed in the body of N. lugens compared with those in ambient CO2 at 4h (P < 0.05). These results showed that there was an instantaneous reaction of N. lugens nymphs to elevated CO2, which indicated N. lugens may enhance stress defense response to future increasing CO2 levels.