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Supplementary Tables 1-4, Figure Legend from Association of Specific Genotypes in Metastatic Suppressor HTPAP with Tumor Metastasis and Clinical Prognosis in Hepatocellular Carcinoma

The phosphatidic acid phosphatase HTPAP has been defined as a metastatic suppressor of hepatocellular carcinoma (HCC), but little is known about its function or potential applications as a prognostic marker. In this study, we analyzed patterns of HTPAP genetic variation and gene expression in 864 patients who underwent HCC resection, assessing these patterns for correlations to tumor metastasis potential. Focusing on two tagSNPs that were selected (+357G/C and +1838A/G), we found that only the +357G/C genotype was significantly associated with HTPAP mRNA and protein expression levels and the probability of metastasis. In an independent cohort of 665 HCC patients, we determined that the +357G/C genotype was associated with shorter time to recurrence and overall survival. Together, these results indicated that the HTPAP tagSNP +357 GG+GC genotypes may influence HCC metastatic potential and clinical prognosis by down-regulating HTPAP expression. Extending these results, a global expression profiling analysis identified 41 genes including the pro-inflammatory genes IL-8 and TLR2 that were significantly overexpressed in the +357 GG+GC group, as possible coregulated markers with HTPAP. Together, our findings identify an HTPAP genotype and associated gene expression pattern that favors metastasis progression and that could be used to predict tumor metastasis and prognosis in HCC patients. Cancer Res; 71(9); 3278–86. ©2011 AACR.

Supplementary Figure 1 from Association of Specific Genotypes in Metastatic Suppressor HTPAP with Tumor Metastasis and Clinical Prognosis in Hepatocellular Carcinoma

Optical clock networks play important roles in various fields, such as precise navigation, redefinition of "second" unit, and gravitational tests. To establish a global-scale optical clock network, it is essential to disseminate time and frequency with a stability of $10^{-19}$ over a long-distance free-space link. However, such attempts were limited to dozens of kilometers in mirror-folded configuration. Here, we take a crucial step toward future satellite-based time-frequency disseminations. By developing the key technologies, including high-power frequency combs, high-stability and high-efficiency optical transceiver systems, and efficient linear optical sampling, we demonstrate free-space time-frequency dissemination over two independent links with femtosecond time deviation, $3\times10^{-19}$ at 10,000 s residual instability and $1.6\times10^{-20}\pm 4.3\times10^{-19}$ offset. This level of the stability retains for an increased channel loss up to 89 dB. Our work can not only be directly used in ground-based application, but also firmly laid the groundwork for future satellite time-frequency dissemination., 27 pages, 13 figures, 2 tables

Objective: To investigate the effect of hepatic differentiation of human bone marrow mesenchymal stem cells (HBMSCs) induced in vitro and transplanted into rats with liver failure via portal vein, and observe the changes of liver function and pathological tissue. Method: After passage to the 6th generation in vitro, the hepatic differentiation was induced by HGFand EGF inducible factors. CCL4 acute liver failure model in rats were established, and randomly divided into 5 groups transplanted with differentiated stem cells via portal vein. These five groups included HGF-differentiated HBMSCs transplantation, EGF-differentiated HBMSCs transplantation, EGF+HGF-differentiated HBMSCs transplantation, non-differentiated HBMSCs transplantation, and non-HBMSCs transplantation. Liver function and pathological changes were detected. Results: Rats models survival, serum albumin, aminotransferase and coagulation indexes were observed at 12 h, 72 h, 7 d, 1 month and 2 months after treatment. The results showed that the survival and albumin, aminotransferase and coagulation function of rats were improved significantly after treatment in HGF-differentiated, EGF-differentiated, EGF+HGF-differentiated and non-differentiated transplantation groups, compared tothe non- HBMSCstransplantation group(P0.05).Pathological changes was ameliorated in the liver of rat models in HGF-, EGF-, EGF+HGF- and non-differentiated transplantation groups, compared to the non- HBMSCs transplantation group. Conclusion: Liver-differentiated BMSCs transplanted into rats with liver failure could effectively improve liver function and survival rate.

Planthoppers are serious rice pests in Asia. Their population resurgence was first reported in the early 1960s, caused mainly by insecticides that indiscriminately killed beneficial arthropods and target pests. The subsequent resurgence involved two mechanisms, the loss of beneficial insects and insecticide-enhanced planthopper reproduction. In this review, we identify two forms of resurgence, acute and chronic. Acute resurgence is caused by traditional insecticides with rapid resurgence in the F1 generation. Chronic resurgence follows application of modern pesticides, including fungicides and herbicides, with low natural enemy toxicity, coupled with stimulated planthopper reproduction. The chemical-driven syndrome of changes leads to later resurgence in the F2 or later generations. Chronic resurgence poses new threats to global rice production. We review findings on the physiological and molecular mechanisms of chronic planthopper resurgence and suggest research directions that may help manage these new threats.

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen) is a major pest insect of rice, wheat, and maize in China and other countries. SBPH not only damage rice plants through sucking plant sap, but also transmits rice virus diseases, for example, striped virus disease (RSV), black streaked dwarf, and maize rough disease virus. Therefore, understanding of pesticide-induced stimulation of reproduction in SBPH is of great significance for the pest management. Our previous study discovered that triazophos (TZP) increased reproduction of SBPH. But the molecular mechanisms are unclear. Here, by using proteomic analysis, we screened and cloned the gene of long chain fatty acid coenzyme A ligase ( FACL ), and silenced FACL to examine influences of TZP on reproduction and glycerin content in SBPH females. In TZP-treated females vs control females, there were 41 differential proteins in 18 pathways related to reproduction, of which 8 were up-regulated and 33 were down-regulated. TZP + dsFACL eliminated TZP-induced stimulation of reproduction of SBPH females (↓about 73.92%) and decreased glycerin content and body weight (↓about 19.93% and 13.62%). TZP + dsFACL treatment led to reduced expression of FACL (↓about 61.88%). FACL is a key gene of TZP-induced increase of reproduction of SBPH.

The widespread use of pesticides in rice has caused the resurgence of the small brown planthopper (SBPH), Laodelphax striatellus. In addition to inciting damage by feeding on phloem cells, SBPH also functions as a vector for rice stripe virus (RSV), which can cause serious yield losses. In general, studies focused on pesticide-induced stimulation of SBPH populations have focused on the insects; little information is available on the impact of pesticides on RSV transmission by SBPH. The present study investigated the effects of two pesticides (validamycin and triazophos) on the fecundity and RSV transmission ability of SBPH. Our results demonstrated that the fecundity of non-viruliferous (naive) or viruliferous SBPH was enhanced by exposure to triazophos at the LC20 and validamycin at 200 mg L− 1. Meanwhile, the increased number of eggs produced by viruliferous females treated with pesticides was larger than those from naive females. Exposure to validamycin caused an increase in horizontal transmission of RSV; furthermore, vertical viral transmission rates of SBPH were significantly increased by exposure to triazophos. The present study provides valuable information for understanding the mechanisms underlying the resurgence of SBPH and subsequent outbreaks of RSV.

PHF7 exhibits male-specific expression in early germ cells, germline stem cells and spermatogonia in insects, and its expression promotes spermatogenesis in germ cells when they are present in a male soma. However, the influence of male-specific PHF7 on female reproductive biology via mating remains unclear. Thus, we investigated the potential impacts of male PHF7, existed in seminal fluid of Nilaparvata lugens (NlPHF7), on fecundity and population growth via mating. Our results revealed that suppressing male NlPHF7 expression by RNAi led to decreases in body weight, soluble accessory gland protein content, arginine content, and reproductive organ development in males, resulting in significant reduction of oviposition periods and fecundity in females, and significant decrease in body weight, fat body and ovarian protein content, yeast-like symbionts abundance, ovarian development and vitellogenin gene expression in their female mating partners. Similarly, suppression of NlPHF7 expression in males mated with the control female reduced population growth and egg hatching rate, but did not influence gender ratio. We infer that NlPHF7 play a role important in stimulating female fecundity via mating. This study provides valuable information by identifying a potentially effective target gene for managing BPH population through RNAi.

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen), is a serious pest insect of rice, wheat, and maize in China. SBPH not only sucks plant sap but also transmits plant disease viruses, causing serious damage. These viruses include rice striped virus disease (RSV disease), black streaked dwarf, and maize rough disease virus. SBPH outbreaks are related to the overuse of pesticides in China. Some pesticides, such as triazophos, stimulate the reproduction of SBPH, but an antibiotic fungicide jinggangmycin (JGM) suppresses its reproduction. However, mechanisms of decreased reproduction of SBPH induced by JGM remain unclear. The present findings show that JGM suppressed reproduction of SBPH (↓ approximately 35.7%) and resulted in the down-regulated expression of glucose dehydrogenase (GDH). GDH- silenced control females (control + dsGDH) show that the number of eggs laid was reduced by 48.6% compared to control females. Biochemical tests show that the total lipid and fatty acid contents in JGM-treated and control + dsGDH females decreased significantly. Thus, we propose that the suppression of reproduction in SBPH induced by JGM is mediated by GDH via metabolic pathways.

Background Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death worldwide. Despite recent advances in imaging techniques and therapeutic intervention for HCC, the low overall 5-year survival rate of HCC patients remains unsatisfactory. This study aims to find a gene signature to predict clinical outcomes in HCC. Methods Bioinformatics analysis including Cox’s regression analysis, Kaplan-Meier (KM) and receiver operating characteristic curve (ROC) analysis and the random survival forest algorithm were performed to mine the expression profiles of 553 hepatocellular carcinoma (HCC) patients from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public database. Results We selected a signature comprising eight protein-coding genes (DCAF13, FAM163A, GPR18, LRP10, PVRIG, S100A9, SGCB, and TNNI3K) in the training dataset (AUC = 0.77 at five years, n = 332). The signature stratified patients into high- and low-risk groups with significantly different survival in the training dataset (median 2.20 vs. 8.93 years, log-rank test P P = 0.004, n = 221, GSE14520). Further multivariate Cox regression analysis showed that the signature was an independent prognostic factor for patients with HCC. Compared with TNM stage and another reported three-gene model, the signature displayed improved survival prediction power in entire dataset (AUC signature = 0.66 vs. AUC TNM = 0.64 vs. AUC gene model = 0.60, n = 553). Stratification analysis shows that it can be used as an auxiliary marker for many traditional staging models. Conclusions We constructed an eight-gene signature that can be a novel prognostic marker to predict the survival of HCC patients.

Previous investigations demonstrated that Chinese wheat cv. Ning7840 confers a high level of broad-spectrum resistance to rusts and powdery mildew diseases at the adult plant stage mediated by a spontaneously occurring hypersensitive reaction-like phenotype (also called lesion mimic, LM). In this study, two EMS-induced mutants of cv. Ning7840, Dlm1 and Dlm2, showed the LM deficiency phenotype. These two mutants were susceptible to powdery mildew. At heading stage when the LM symptoms appear on the leaves of Ning7840, the RNAs were harvested from the flag leaves of Dlm1, Dlm2 and Ning7840 leaves and were sequenced to decipher the molecular mechanisms underlying LM phenotypes. The results suggest that the diterpenoid-related stress response acts as an indirect regulator of the LM phenotype and may be associated with broad-spectrum resistance in Ning7840. Oxidative phosphorylation, proteasome, and chlorophyll were involved in the suppression of the LM expression in the Dlm mutants. These results provide new insight into the molecular mechanism of LM and broad-spectrum resistance in wheat.

The brown plant hopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major pest affecting rice in Asia, and outbreaks of this pest are closely linked to pesticide-induced stimulation of reproduction. Therefore, the BPH is a classic example of a resurgent pest. However, the effects of different genes on the regulation of pesticide-induced reproductive stimulation in the BPH are unclear. In this study, the regulatory effects of acyl-coenzyme A oxidase (ACO) on the reproduction and biochemistry of the BPH were investigated with gene silencing. The number of eggs laid per female by triazophos (TZP)+dsACO BPH females was significantly lower than those of TZP-treated (without ACO silencing) or TZP+GFP females (negative control), with the number of eggs decreasing by 30.8% (from 529.5 to 366.3) and 32.0% (from 540.5 to 366.3), respectively. The preoviposition period, oviposition period, and longevity of the TZP-treated females were also influenced by dsACO treatment. Additionally, the amounts of crude fat, protein, and some fatty acids (oleic acid, palmitic acid, linoleic acid, stearic acid, and myristoleic acid) in TZP+dsACO females were significantly lower than in TZP-treated females. Thus, ACO is one of the key genes regulating the TZP-induced stimulation of reproduction in BPH females.

The antibiotic jinggangmycin (JGM) is mainly used in controlling the rice sheath blight, Rhizoctonia solani, in China. JGM also enhances reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål). To date, however, molecular mechanisms of the enhancement are unclear. Our related report documented the influence of foliar JGM sprays on ovarian protein content. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) protocols to analyze ovarian proteins of BPH females following JGM spray (JGM-S) and topical application (JGM-T). We recorded changes in expression of 284 proteins (142↑ and 142↓) in JGM-S compared to the JGM-S control group (S-control); 267 proteins were differentially expressed (130↑ and 137↓) in JGM-T compared to the JGM-T control group (T-control), of which, 22 proteins were up-regulated in both groups. Comparing the JGM-S to the JGM-T group, 114 proteins were differentially expressed (62↑ and 52↓). Based on the biological significance of fatty acids, pathway annotation and enrichment analysis, we designed a dsRNA construct to silence a gene encoding fatty acid synthase (FAS). FAS was more highly expressed in JGM-S vs S-control and JGM-S vs JGM-T groups. The dsFAS treatment reduced fecundity by about 46% and reduced ovarian and fat body fatty acid concentrations in JGM-S-treated females relative to controls. We infer FAS provides critically needed fatty acids to support JGM-enhanced fecundity in BPH.

Hexokinase is a rate-limiting enzyme that plays pivotal roles in glucose homeostasis and energy metabolism via glucose (Glc) phosphorylation and Glc signaling mediation. Previous investigations have revealed the modulatory role of Hexokinase (Hex) genes involved in proper glucose regulation during insect diapause and embryo development, whereas whether it functions in insect fecundity remains largely unknown. We aimed to explore the relationship between Triazophos (TZP)-induced Hex-1 and fecundity of female Nilaparvata lugens. In this study, Hex-1 expression were characterized at different developmental stages and in various tissues of N. lugens, with the highest expression registered in brain tissues and 5th instar nymph. The present findings indicated that TZP + dsHex-1 silencing significantly reduced protein synthesis, including the fat body and ovarian protein content of female adults. Meanwhile, the glycometabolism with respect to the soluble sugar, trehalose and glucose content in female adults were strikingly influenced as a result of Hex-1 knockdown. The relative transcript level of Hex-1, vitellogenin (NlVg) and vitellogenin receptor (NlVgR) considerably decreased in TZP + dsHex-1 treated females compared to TZP and TZP + dsGFP-treated groups. More importantly, TZP + dsHex-1 silencing led to reduced number of eggs laid and vitellogenin (Vg) accumulation as well as retarded ovary development compared with TZP-treated and TZP + dsGFP-treated groups. Taken together, it is proposed that Hex-1 implicates in N. lugens fecundity by exerting profound effects on glycometabolism, protein sythesis and NlVg expression.

Aside from their intended actions, fungicides can drive pest insect outbreaks due to virtually continuous use and pest evolution. Small brown planthopper (SBPH), Laodelphax striatellus, outbreaks occurred recently in many provinces in China, with devastating rice losses. Because exposure to the fungicide jinggangmycin (JGM) increased reproduction of the brown plant hopper, Nilaparvata lugens, via its influence on fatty acid synthase, we posed the hypothesis that JGM and carbendazim (CBM) influence SBPH reproduction via their influence on enzymes involved in other aspects of lipid metabolism. Exposure to the fungicide CBM stimulated SBPH reproduction (egg-laying up by 78%) and to another fungicide, JGM, led to decreased egg-laying (down by 47.3%). These inverse effects are mediated by down-regulated expression of l-3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) in JGM-treated females and up-regulated expression of hydroxysteroid dehydrogenase-like protein 2-like (HSD) in CBM-treated females. RNAi knockdown of, separately, LCHAD and HSD led to reduced egg-laying (down by 52% for dsLCHAD and by 73% for dsHSD). dsLCHAD, dsHSD, and JGM treatments also led to severely reduced ovarian development in experimental SBPH, with shorted and thinned valvula and lack of egg cells in ovaries. Valvula of CBM-treated females enlarged, with banana-shaped eggs in ovaries. These data strongly support our hypothesis.

The rice leaffolder (RLF), Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae), and the white-backed rice planthopper (WBPH), Sogatella furcifera Horváth (Hemiptera: Delphacidae), are major insect pests in China and several other Asian countries. These two pests commonly occur simultaneously or in a temporal sequence. Thus, the investigation of the effect of complex infestations or temporal sequence infestations by these pests on rice yield has a practical significance for the control of these pests. The present study comprised experiments with the following four different variables in potted rice at the tillering stage: single pest species infestation, complex infestation, complete combination infestation and temporal sequence infestation (C. medinalis infestation prior to S. furcifera and S. furcifera infestation prior to C. medinalis). The results showed that the four infestations resulted in a significant decrease in 1000-grain weight (1000GW) and rate of yield loss (RYL) but an increase in blighted grain rate (BGR), with a significant positive correlation with the infestation density. However, the influences of the complex infestation, complete combination infestation or sequence infestation on the 1000GW, BGR and RYL were greater than those of the single pest species infestations but did not have addition effects, i.e., the effects of the complex infestation and combination infestation or sequence infestation on the 1000GW, BGR and RYL were less than the additive effects of the two single pest species infestations at the same densities. In the condition of the same total infestation pressure, no significant differences in the 1000GW, BGR and RYL were found between C. medinalis infestation prior to S. furcifera and S. furcifera infestation prior to C. medinalis as well as between the sequence infestation and the complex infestation.

Liver cancer remains one of the most lethal malignancies worldwide. We sequenced the complete mitochondrial genome of a liver cancer HA22T cell line. The sequenced mitogenome of 16,603 bp will aid in the use of the HA22T cell line for liver cancer study.

The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH), Nilaparvata lugens Stal. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.

Three planthopper species, the brown planthopper (BPH), Nilaparvata lugens Stål, the white-backed planthopper (WBPH), Sogatella furcifera Horvath, and the small brown planthopper (SBPH), Laodelphax striatella Fallén, often co-occur in rice grown regions of China. The present investigation examined effects of two pesticides, triazophos (TZP) and jinggangmycin (JGM) (a fungicide), on reproduction of BPH, WBPH, and SBPH. The results showed that TZP stimulated the fecundity of the three planthopper species. Interestingly, JGM stimulated the fecundity of BPH but suppressed the fecundity of WBPH. In addition, TZP and JGM had a significant effect on the preoviposition period (PVD), the oviposition period (OPD), and the longevity of adult females (LAF) of BPH and WBPH. Based on these findings, to avoid resurgence occurrence of planthoppers, we suggest that the application of TZP should be banned in rice fields, that JGM should be used to control rice sheath blight at the early growth stages of rice (with WBPH occurrence and without BPH occurrence).

The brown planthopper (BPH) is a devastating pest of rice throughout Asia. In this paper we document the BPH biogeographic range expansion in China over the 20-year period, 1992 to 2012. We posed the hypothesis that the range expansion is due to a syndrome of adaptations to the continuous presence of agricultural chemicals (insecticides and a fungicide) over the last 40 years. With respect to biogeography, BPH ranges have expanded by 13% from 1992 to 1997 and by another 3% from 1997 to 2012. In our view, such expansions may follow primarily from the enhancing effects of JGM, among other agricultural chemicals, and from global warming. JGM treatments led to increased thermotolerance, recorded as decreased mortality under heat stress at 40 ± 1 °C (down from 80% to 55%) and increased fecundity (by 49%) at 34 °C. At the molecular level, JGM treatments led to increased abundances of mRNA encoding Acetyl Co-A carboxylase (Acc) (up 25%) and Hsp70 (up 32%) in experimental BPH. RNAi silencing of Hsp70 and Acc eliminated the JGM effects on fecundity and silencing Hsp70 reduced JGM-induced thermotolerance. Integrated with global climate change scenarios, such syndromes in pest insect species have potential for regional- and global-scale agricultural disasters.

Potassium (K) is a key component of plant nutrition, significantly influencing crop growth. Levels of this nutrient in plants can also influence a number of pest infestations. The brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), is an important pest of rice in Asia. In this study, we examined K contents in rice grown in hydroponic solution, and its relationship to the fecundity and expression of the vitellogenin (Nlvg) gene of N. lugens which was reared on the rice. Our findings indicated that K contents in rice increased with the increasing K concentration within the hydroponic solution, but reduced at the highest K concentration (160 mg/L). The number of eggs laid by N. lugens which was reared on the rice varied significantly with K concentration, and increased by 0.12 and 0.22 fold under 20 mg/L and 160 mg/L K level compared to that of the control (40 mg/L), decreasing by 0.57 fold under 0 mg/L K. Nlvg mRNA expression increased 1.17 and 1.94 fold in individuals which were reared on rice grown in 20 mg/L and 160 mg/L K level, compared to that of the control before mating; and by 3.36 and 2.97 after mating, respectively. However, Nlvg mRNA expression fold decreased by 0.99 under 0 mg/L K level before mating and 0.91 after mating. The variation of eggs may be attributed to the change of Nlvg mRNA expression, because there was a positive correlation between the eggs and Nlvg mRNA expression. These results demonstrated low (20 mg/L) and highest K levels (160 mg/L) in hydroponic solution showed the lower K level in plants than the control, which facilitated the fecundity of N. lugens. The study of the effects of K levels on the fecundity should have significance for insect control.

Climate change is likely to have marked ecological effects on terrestrial ecosystems, including the activities of insect pests. Most attention has focused on the increasing geographical ranges of pests; however, if extrinsic factors enhance their thermotoler- ance, populations may express increased voltinism and longer daily and annual activ- ity periods. These changes in pest populations have the potential for severe consequences, including increased crop losses and decreased food security at the glo- bal level. The brown planthopper (BPH) Nilaparvata lugens Stal (Hemiptera: Delphaci- dae) is a serious pest of rice crops in temperate and tropical regions of Asia. It is often present in rice microclimates at temperatures close to its maximum thermotolerance. Recent BPH outbreaks in tropical Asia are considered to be associated with excess use of pesticides and increasing temperature. This study tested whether exposure to suble- thal concentrations of triazophos (tzp), an insecticide widely used in Asian rice pro- duction, enhances thermotolerance of BPH. Tzp exposure (40 ppm at 40 °C) significantly decreased mortality (from 94% in controls to 50% at 48 h post-treatment) and increased lethal mean time (LT50) of adults by 17.2 h. To investigate the underly- ing molecular mechanism of this tzp-enhanced thermotolerance, we selected Hsp70 and Arginine kinase (Argk) for detailed study. Transcripts encoding both proteins in third-instar nymphs and brachypterous adult females were up-regulated, compared with controls, after exposure to tzp. RNAi silencing of both genes demonstrated that Hsp70 and Argk are essential for survival and tzp-increased thermotolerance. We pro- pose that tzp induces thermotolerance in BPHs by increasing the expression of genes that act in cell protection mechanisms. The significance of our proposal relates to the importance of understanding the influence of sublethal concentrations of insecticides on pest biology. In addition to its influence on thermotolerance, tzp also enhances BPH reproduction. We infer that exposure to a pesticide stressor can produce cross- tolerance, that is, increased tolerance to one stressor also increases tolerance to other stressors, including temperature. Aside from needing a better understanding of these effects in nature and in other pest/cropping systems, we suggest that pest management programmes can be improved with better understanding of the influences of stressors, including increased environmental temperatures and sublethal concentrations of insec- ticides, on pest biology.

The brown planthopper (BPH), Nilaparvata lugens Stal, is a serious rice pest throughout Asia. Recent outbreaks of N. lugens populations were mainly associated with the overuse of pesticides and resistance to insecticides. Warmer global temperatures that are associated with anthropogenic climate change are likely to have marked ecological effects on terrestrial ecosystems. However, the effects of elevated CO2 concentrations on the biochemical, physiological and nutrient quality of transgenic Bt rice that has been treated with pesticides and on the control efficacy of the pesticides are not understood. The present study investigated changes in soluble sugar content, free amino acid levels, oxalic acid levels, flavonoids levels, and triazophos residues in transgenic Bt rice (TT51) and the control efficacy of triazophos for N. lugens following triazophos foliar spray under conditions of elevated CO2 (eCO2). Our findings showed that the soluble sugar content of TT51 treated with triazophos under eCO2 was significantly higher than that under ambient CO2 (aCO2) and also higher than that of the non-transgenic parent (MH63) under aCO2. However, the results for free amino acid levels were the opposite of those for soluble sugar levels. The oxalic acid and flavonoid contents of rice plants significantly decreased with increases in triazophos concentration, CO2 concentration, and days after treatment (DAT). The oxalic acid and flavonoid contents of TT51 treated with triazophos under eCO2 were significantly lower than those under aCO2 and also lower than those of MH63 under aCO2. The residue concentration of triazophos varied with CO2 concentration, rice variety, and DAT. The residues in TT51 treated with 80 ppm of trizaopos under eCO2 were significantly lower than those under aCO2 and those in MH63 under aCO2. The survival rate of nymphs N. lugens in TT51 under eCO2 was significantly higher than that under aCO2 and that in MH63 under aCO2 at 1 DAT or 15 DAT after the release of 2nd instars nymphs. These findings indicated that (1) for TT51, triazophos reduced the resistance of rice plants to N. lugens with an elevated CO2 concentration, as N. lugens consumed more phloem sap on TT51 plants; (2) triazophos dissipation in TT51 under eCO2 was significantly faster than that under aCO2 and that in MH63 under aCO2; (3) the control efficacy of triazophos for N. lugens significantly decreased under eCO2. The present findings provide important information for integrated pest management among transgenic varieties.

Effects of transgenic Bt rice on non-target pests following pesticide applications plays an important role in evaluating the ecological safety of transgenic rice. Changes of the life history parameters of laboratory population of Nilaparvata lugens feeding on transgenic Bt rice T2A-1 and its parental line cv. MH63 as the control, and the contents of oxalic acid and soluble sugar following three pesticides (triazophos, jinggangmycin and chlorantraniliprole) treatments were investigated in this paper. Results showed that the population parameters of N. lugens and the physiological and biochemical parameters of rice did not differ significantly between T2A-1 and MH63 without pesticide application. But, the emergence rate (ER), the nymphal number of next generation (NN), and the index of population trend (IP) on T2A-1 treated with 10 mg L −1 of triazophos were significantly higher than those on MH63, respectively, while the hatchability (HB) on T2A-1 treated with the three pesticides were obviously lower than those on MH 63. Furthermore, the content of oxalic acid in MH63 treated with 20 mg L −1 triazophos and, 40 and 80 mg L −1 chlorantraniliprole was significantly higher than that in T2A-1, while the content of soluble sugar in T2A-1 treated with 400 mg L −1 jinggangmycin was significantly higher than that in MH63. The results above indicated that triazophos in a low dose could cause a more favor of T2A-1 to population growth of N. lugens compared with MH 63.

The new insecticide indoxacarb was recently developed with high insecticidal activity in the control of lepidopteran pests, and recent findings demonstrated that indoxacarb also significantly suppressed the fecundity of Nilaparvata lugens Stal (Hemiptera: Delphacidae) populations, a serious insect pest of rice in Asia. The current study investigated the total protein, RNA, and DNA contents in the ovaries and fat bodies of N. lugens adult females after treatment with the insecticide indoxacarb. The results demonstrated that all of the tested concentrations of indoxacarb caused a significant decrease in the protein and RNA contents in the ovaries and fat bodies of the adult females that developed from nymphs fed indoxacarb-treated rice plants compared with those from nymphs fed untreated rice plants, while the DNA contents did not significantly decrease except at 5 days among all of the tested days after emergence. In general, the protein and RNA contents in both the ovaries and the fat bodies decreased linearly with the concentration of indoxacarb applied. There was a significant negative correlation between the total protein and RNA contents and the concentrations of indoxacarb, showing that the total protein and RNA were more sensitive to indoxacarb than was the DNA, and that the changes in RNA contents in turn influence protein synthesis in both the ovaries and fat bodies. The RNA-to-protein ratios in both the ovaries and fat bodies were consistently 0.2 in all of the treatment groups and in the control groups.

The rice leaffolder, Cnaphalocrocis medinalis Guenee (Lepidoptera: Pyralidae), is a rice pest that is prone to outbreaks when high levels of nitrogen (N) fertilizer are applied to rice. The larvae feed by scraping the mesophyll cells of leaves, which are mainly composed of chloroplasts containing significant levels of elemental magnesium (Mg). To determine the cause of N fertilizer-induced rice leaffolder outbreaks and the effects of Mg and Mg uptake on the rice leaffolder, the changes in the life history traits and biochemistry of C. medinalis larvae feeding on rice leaves treated with different Mg and N concentrations were investigated in this study. The results showed that foliar treatment using different Mg concentrations under hydroponic culture conditions significantly decreased the developmental duration (days) (DD) of the larvae and pupae but did not influence the pupation rate or adult emergence rate. The number of eggs laid by adult females and the protein content and total sugar content in the larvae and pupae significantly increased with increasing Mg concentrations. Under soil culture conditions, the DD for the larvae and number of eggs laid by adult females significantly increased with increasing nitrogen fertilizer levels, but the applied fertilizer did not influence the DD in the pupae, pupation rate, or adult emergence rate. In contrast, the DD for adults decreased with increased fertilizer levels. In hydroponic culture, Mg in the culture solution and Mg foliar sprays significantly increased the Mg content in rice leaves. In soil cultures, the use of N fertilizer and Mg foliar spray did not significantly increase the leaf Mg content, except at a concentration of 12.5 g/L, indicating that the leaf Mg content may be related to the root uptake of Mg.

The brown planthopper (BPH), Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), is a long-distance migratory insect pest in temperate eastern Asia and is a classical resurgent rice pest that is induced by insecticides. Knowledge of the effect of insecticides on the flight capacity of insects is needed to understand their migration, which may help to improve the management of insect pests. Our previous study demonstrated that some insecticides enhance the flight capacity of BPH. However, the effect of insecticides on the flight muscle of BPH is not well understood. Thus, the present study examined the effect of triazophos, a classical insecticide that induces the resurgence of BPH, on the ultrastructure of macropterous females using transmission electron microscopy. The results showed that the diameter of myofibrils one and two days after adult female emergence (DAE) following treatment with 40 ppm triazophos was significantly greater than that of the control, increasing by 31.4 and 21.5%, respectively. In addition, the length of the sarcomeres at two and three DAE was significantly greater than that of the control, increasing by 73.8 and 50.8%, respectively. The percentage of mitochondrial volume in the muscle fibres at one and two DAE was 146.2 and 67.7% greater than that of the control, respectively. These findings show that the mechanisms of triazophos-induced enhancement of the flight capacity of BPH involve changes in the ultrastructure of the flight muscle.

The brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), is a classic example of a pest that experiences insecticide-induced fecundity. The insecticides indoxacarb and chlorantraniliprole were recently developed with high insecticidal activity in the control of rice yellow stem borer, Tryporyza incertulas (walker) and rice leaffolder, Cnaphalocrocis medinalis (Guenee). We examined the changes in the fecundity of N. lugens , the vitellogenin ( Nlvg ) gene expression and the vitellin contents of adult females exposed to these new insecticides. Our findings showed that indoxacarb and chlorantraniliprole significantly suppressed the fecundity of the females and the expression of Nlvg mRNA. Treatment with 10, 20, 40 and 80 mg/L indoxacarb decreased Nlvg mRNA expression by 35.4%, 74.4%, 79.6% and 61.9%, respectively, and treatment with 7.5, 15, 30, 60 and 80 mg/L chlorantraniliprole decreased Nlvg mRNA expression by 79.3%, 40.2%, 40.0%, 79.8% and 51.8%, respectively. The vitellin contents in both the ovaries and fat bodies of the adult females also decreased, indicating that the two insecticides suppressed the synthesis of vitellin in the fat bodies and the transfer of vitellin to the ovaries. This decrease in the vitellin contents of the ovaries may be attributed to the low Nlvg expression, as there was a positive correlation between the vitellin content and the expression of this gene, indicating that these insecticides influenced the fecundity of N. lugens by affecting the expression of Nlvg mRNA. The study of the effects of indoxacarb and chlorantraniliprole on fecundity should have significance for the wide usage in rice plants.

In our previous study with the brown planthopper (BPH), Nilaparvata lugens, triazophos (tzp) treatments led to substantial up-regulation of a male spermatogenesis-associated protein 5-like gene (NlSPATA5) compared to untreated controls. Mating with tzp-treated males significantly increased fecundity (as numbers of eggs laid), relative to females mated with untreated males. Because SPATA5 acts in mammalian sperm development and is expressed in testes, we posed the hypothesis that NlSPATA5 occurs in BPH seminal fluid and it operates in fecundity via mating. We tested the hypothesis by investigating the influence of suppressing NlSPATA5 expression in BPH males on fecundity. Reduced expression of NlSPATA5 led to decreased male accessory gland protein content and reproductive system development compared to controls. These changes in males led to prolonged pre-oviposition periods and decreased fecundity in females. For both genders, we recorded no difference in the body weight, oviposition periods, and longevity compared to controls. NlSPATA5 suppression in males also led to decreased fat body and ovarian protein content, yeast-like symbionts abundance and ovarian development as well as vitellogenin gene expression in their mating partners. We infer that increased NlSPATA5 expression may be one molecular mechanism of tzp-driven reproduction and population increases in BPH.

Using bulk segregant analysis (BSA) coupling with RNA-seq and DNA markers identified a potentially novel nitrogen-dependent lesion mimic gene Ndhrl1 on 2BS in wheat. Lesion mimic (LM) refers to hypersensitive reaction-like (HRL) traits that appear on leaf tissue in the absence of plant pathogens. In a wheat line P7001, LM showed up on the leaves under the 0 g nitrogen (N) treatment, but disappeared when sufficient N was supplied, suggesting that LM is N-responsive and N dosage dependent. Using BSA strategy together with RNA-seq and DNA markers, we identified an N-dependent LM gene (Ndhrl1) and mapped it to the short arm of chromosome 2B using an F5 recombinant inbred population developed from the cross of P7001 × P216. The putative gene was delimited into an interval of 8.1 cM flanked by the CAPS/dCAPS markers 7hrC9 and 7hr2dc14, and co-segregated with the dCAPS marker 7hrdc2. This gene is most likely a novel gene for LM in wheat based on its chromosomal location. Further analysis of RNA-seq data showed that plant–pathogen interaction, nitrogen metabolism, zeatin biosynthesis and plant hormone signal transduction pathways were significantly differentially expressed between LM and non-LM lines.

Previous studies have demonstrated that the agricultural antibiotic jinggangmycin (JGM) stimulates reproduction in the brown planthopper Nilaparvata lugens Stal and that the stimulation of brown planthopper reproduction induced by JGM is regulated by the fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) genes. However, a key issue in the stimulation of reproduction induced by pesticides involves the growth and development of oocytes. Therefore, the present study investigated oocyte changes via transmission electron microscopy (TEM) and changes in hormone levels (juvenile hormones (JH) and 20-hydroxyecdysone (20 E)) in JGM-treated females. TEM observations showed that the size of the lipid droplets in the oocytes of JGM-treated females, compared with those in the oocytes of the control females, significantly reduced by 32.6 and 29.8% at 1 and 2 d after emergence (1 and 2 DAE), respectively. In addition, the JH levels of JGM-treated females at 1 and 2 DAE were increased by 49.7 and 45.7%, respectively, whereas 20 E levels decreased by 36.0 and 30.0%, respectively. We conclude that JGM treatments lead to substantial changes in lipid metabolism, which are directly and indirectly related to stimulation of reproduction of brown planthopper together with our previous findings.

The antibiotic jinggangmycin (JGM) is an agrochemical product widely used in China for controlling rice sheath blight, Rhizoctonia solani. Unexpectedly, it stimulates reproduction in the brown planthopper (BPH), Nilaparvata lugens (Stål). However, the underlying molecular mechanisms of the stimulation are unclear. The present investigation demonstrates that adipose triglyceride lipase (Atgl) is one of the enzymes involved in the JGM-stimulated reproduction in BPH. Silence of Atgl in JGM-treated (JGM + dsAtgl) females eliminated JGM-stimulated fecundity of BPH females. In addition, Atgl knockdown significantly reduced the protein and glycerin contents in the ovaries and fat bodies of JGM + dsAtgl females required for reproduction. We conclude that Atgl is one of the key enzymes responsible for JGM-stimulated reproduction in BPH.