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Background Sugarcane is the most important sugar crops in the world. Like other crops, sugarcane is also suffered from herbivorous insect attack. The oriental armyworm Mythimna separata is a devastating pest of various crop in northeast Asia, outbreak of this pest resulted in substantially yield loss including sugarcane. However, the plant defense response situation is widely acquisition in model crops, but little information about how sugarcane plants defend themselves against this herbivore at the molecular and biochemical levels is studied. Results We combined transcriptome and metabolomics analysis to investigate the changes in gene expression and metabolic processes that occurred in sugarcane plants after continuous feeding by M. separata larvae for 12, and 24 h. We identified 13 662 genes and 55 metabolites that were differentially regulated in sugarcane plants fed upon by M. separata. The genes involved in phytohormones, transcription factors, and kinase-related were activated and the metabolism compounds such as carbohydrate, amino acid, ferulic substances and glutathione were detected regulated in sugarcane defense response. Comparable analyses showed a close correspondence relationship among pathways of phenylalanine metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis in transcript and metabolite profiles. Furthermore, a bioassay experiment was conducted to test the influence of up-regulated metabolites on M. separata growth and found chlorogenic acid showed lethal effect. Conclusion The results of our study greatly enhanced understanding on sugarcane induced defense response mechanism against herbivore infestation at the transcriptional and metabolic levels. Also make contributions to provide clues for development of green pest control method. This article is protected by copyright. All rights reserved.

The purpose of this study was to investigate the role of a human lens microRNA (miR-497-5p) in regulating epithelialmesenchymal transition (EMT) under the control of transforming growth factor beta (TGF-β). A microRNA array was used to evaluate the microRNA profiles of untreated and TGF-β-treated human lens epithelial cells in culture. This showed that TGF-β treatment led to the upregulation of 96 microRNAs and downregulation of 39 microRNAs. Thirteen microRNAs were predicted to be involved in the pathogenesis of posterior capsule opacification (PCO). Meanwhile, overexpression of miR-497-5p suppressed cell proliferation and EMT 48 h post-transfection, and inhibition of miR-497-5p accelerated cell proliferation and EMT. Treatment with TGF-β inhibited the expression of miR-497-5p, but not cell proliferation. miR-497-5p was also found to regulate the level of CCNE1 and FGF7, which are reported to be actively involved in EMT. CCNE1 and FGF7 were bona fide targets of miR-497-5p. The results suggest that miR-497-5p participates in the direct regulation of lens epithelial cell EMT and is regulated by TGF-β. miR-497-5p may be a novel target for PCO therapy.

Bemisia tabaci is one of notorious agricultural insect pests in China, and the strategies of management largely depend on application of insecticides. In order to assess levels of resistance in field populations of B. tabaci to six insecticides including abamectin, cyantraniliprole, pymetrozine, imidacloprid, chlorpyrifos and bifenthrin, we monitored the susceptibility to all tested insecticides in five field populations across China and the results indicated that field populations of B. tabaci have developed various levels of resistance to each chemical agent. Furthermore, para-type voltage gated sodium channel mutations (L925I and T929V) and acetylcholinesterase ace1 mutation (F331W) were confirmed, and expression levels of CYP6CM1, CYP4C64, GSTd7 and ABCG3 were detected for investigating mechanisms of imidacloprid resistance in the five field-collected populations. The results showed that, in all tested populations, frequencies of F331W were 100%, and the frequencies of the L925I and T929V were in the range of 28.5 to 47.0% and 11.0 to 53.5%, respectively. Moreover, CYP6CM1 and CYP4C64 were significantly overexpressed in two tested populations, respectively, and GSTd7 was significantly overexpressed in one population. No overexpression of ABCG3 was observed in all the populations. Above results provided valuable insight into the current status of insecticide resistance and could be contributed to design strategies of management for B. tabaci.

Silicon (Si) is considered to be a plant growth and development regulator element as well as provide the regulatory response against various biotic stressors. However, the potential mechanism of Si enhancement to regulate plant disease resistance remains to be studied. Therefore, the current study evaluated the effects of Si application on the performance of sugarcane against Xanthomonas albilineans (Xa) infection. Si was applied exogenously (0, 3.85 and 7.70 g Si/kg soil) and the results show that plant height, stem circumference and leaf width of siliconized sugarcane have been improved, which effectively reduced the disease index (0.17–0.21) and incidence (58.2%−69.1%) after Xa infection. Lowest values of MDA (348.5 nmol g−1 FW) and H2O2 (3539.4 mmol/L) were observed in 7.70 g Si/kg soil followed by in 3.85 g Si/kg soil (MDA: 392.6 nmol g−1 FW and H2O2: 3134.6 mmol/L) than that of the control. Whereas, PAL enzyme activity (50.8 mmol/L), JA (230.2 mmol/L) and SA (2.7 ug mL−1) contents were significantly higher in 7.70 g Si/kg soil followed by in 3.85 g Si/kg soil (PAL: 46.3 mmol/L, JA: 182.7 mmol/L and SA: 2.4 ug mL−1) as compared to control. The lower MDA, H2O2 level and higher enzymatic activities were associated with the highest expression levels of their metabolic pathway associated genes i.e., ShMAPK1, ShLOX, ShPAL, ShAOS, ShAOC, ShC4H, ShCAT, Sh4CL and ShNPR1 (22.08, 15.56, 10.42, 3.35, 2.54, 2.14, 1.82, 1.67 and 1.22 folds, respectively) in 7.70 g Si/kg soil as compared to other experimental units and control. Overall, the results of current study indicates that siliconized sugarcane more actively regulates disease resistance through modulation of growth and MDA, H2O2, SA and JA associated metabolic pathways.

Cyantraniliprole is a novel anthranilic diamide insecticide with significant efficacy against Bemisia tabaci, an important pest insect worldwide. In this study, we conducted reversion and selection work and genetic analysis, and determined cross-resistance spectrum and synergism of cyantraniliprole resistance based on the reported population, SX population, of B. tabaci collected from Shanxi Province, China. Compared with a susceptible strain (MED-S), SX population, the field-evolved cyantraniliprole-resistant population exhibited 26.4-fold higher resistance to cyantraniliprole. In SX, a sharp decline of cyantraniliprole resistance was shown in the absence of selection. Another tested strain, SX-R, was established from SX population after successive selection with cyantraniliprole and recently developed 138.4-fold high resistance to cyantraniliprole. SX-R had no cross-resistance to abamectin, imidacloprid, thiamethoxam, sulfoxaflor, or bifenthrin. Genetic analysis illustrated that cyantraniliprole resistance in SX-R was autosomally inherited and incompletely dominant. Additionally, piperonyl butoxide (PBO) significantly inhibited cyantraniliprole resistance in the SX-R strain. In conclusion, the selection of SX with cyantraniliprole led to high resistance to cyantraniliprole which is incompletely dominant and no cross-resistance to several common types of insecticides. Enhanced oxidative metabolism is possibly involved in the resistance of SX-R, yet target-site resistance could not be excluded.

Herbivore attack leads to enhanced production of defensive compounds to mount anti-herbivore defense in plants via activation of the jasmonate signaling pathway. On the other hand, some herbivores can eavesdrop on plants defense signaling and up-regulate their cytochrome P450 genes to increase detoxification of defensive compounds. However, the ecological risk of eavesdropping on plant defense signaling is largely unknown. In this study, we examined the induction of cytochrome P450s by methyl jasmonate (MeJA) and its consequence on the toxicity of aflatoxin B1 (AFB1) to Helicoverpa armigra larvae. The results show that MeJA applications either in a diet or volatile exposure enhanced the toxicity of AFB1 to the larvae. RNA sequences analysis revealed that cytochrome P450 CYP6AE19 was highly induced when MeJA was applied with AFB1. In addition, HaGST encoding glutathione-S-transferase that mainly transforms aflatoxin B1 exo-8,9-epoxide to aflatoxin B1 exo-8,9-glutathione was also induced. RNA interference of CYP6AE19 via injecting a double-stranded RNA decreased mortality of larvae exposed to AFB1; while injecting a double-stranded RNA of HaGST increased larval mortality. Furthermore, a protein model was generated and a subsequent docking simulation for AFB1 suggests the bioactivation as a major mechanism of AFB1. This study provides evidence that MeJA increased larval mortality of H. armigera via induction of CYP6AE19 that can bioactivate AFB1.

Glutamate-gated chloride channels (GluCls) mediate inhibitory synaptic transmission in invertebrate nervous systems, and only one GluCl gene has been found in insects. Therefore, insect GluCls are one of the major targets of insecticides including avermectins. In the present study, a 1347 bp full-length cDNA encoding a 449-amino acid protein (named MsGluCl, GenBank ID: MK336885) was cloned from the oriental armyworm, Mythimna separata, and characterized two alternative splicing variants of MsGluCl. The protein shares 76.9–98.6% identity with other insect GluCl isoforms. Spatial and temporal expression analysis revealed that MsGluCl was highly expressed in the 3rd instar and adult head. Dietary ingestion of dsMsGluCl significantly reduced the mRNA level of MsGluCl and decreased abamectin mortality. Thus, our results reveal that MsGluCl could be the molecular target of abamectin and provide the basis for further understanding the resistance mechanism to abamectin in arthropods.

In order to understand the genetic diversity and structure within and between the genera of Saccharum and Erianthus, 79 accessions from five species (S. officinarum, S. spontaneum, S. robustum, S. barberi, S. sinense), six accessions of E. arundinaceus, and 30 Saccharum spp. hybrids were analyzed using 21 pairs of fluorescence-labeled highly poloymorphic SSR primers and a capillary electrophoresis (CE) detection system. A total of 167 polymorphic SSR alleles were identified by CE with a mean value of polymorphic information content (PIC) of 0.92. Genetic diversity parameters among these 115 accessions revealed that Saccharum spp. hybrids were more diverse than those of Saccharum and Erianthus species. Based on the SSR data, the 115 accessions were classified into seven main phylogenetic groups, which corresponded to the Saccharum and Erianthus genera through phylogenetic analysis and principle component analysis (PCA). We propose that seven core SSR primer pairs, namely, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, SMC24DUQ, mSSCIR3, and mSSCIR43, may have a wide appicability in genotype identification of Saccharum species and Saccharum spp. hybrids. Thus, the information from this study contibites to manage sugarcane genetic resources.

The oriental armyworm Mythimna separata is a serious polyphagous pest in China and there are major efforts to control this pest. In the present study, an RNA-Seq method was used to explore transcriptome data of M. separata and identify the responses of genes to chlorantraniliprole. Sequencing and de novo assembly yielded 134,533 transcripts that were further assembled into 77,628 unigenes with an N50 length of 2165 bp. A total of 76 unigenes encoding insecticide targets were identified. Furthermore, 62 cytochrome P450s, 34 glutathione S-transferase (GSTs)and 64 carboxylesterase (CCEs) were curated to construct phylogenetic trees. In addition, we identified 647 the differentially expressed genes following treatment with chlorantraniliprole. The pathways of calcium signaling was identified as response to the pesticide The transcriptome data we generated represents a comprehensive genomic resource for further studies focused on control of M. separata. The response of genes to chlorantraniliprole treatment will elucidate the molecular mechanisms of insecticide resistance and allow for the development of new chemical pesticides to control this pest.

The β-adrenergic-like octopamine receptor (OA2B2), which binds the biogenic amine octopamine, belongs to the class of G-protein coupled receptors and significantly regulates many physiological and behavioral processes in insects. In this study, the putative open reading frame sequence of the MsOA2B2 gene in Mythimna separata was cloned, the full-length complementary DNA was 1191 bp and it encoded a 396-amino acid protein (GenBank accession number MN822800). Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. Real-time quantitative polymerase chain reaction of spatial and temporal expression analysis revealed that the MsOAB2 gene was expressed in all developmental stages of M. separata and was most abundant in egg stages and second and fourth instars compared with other developmental stages, while the expression level during the pupal stage was much lower than that at the other stages. Further analysis with sixth instar M. separata larvae showed that the MsOA2B2 gene was expressed 1.81 times higher in the head than in integument and gut tissues. Dietary ingestion of dsMsOA2B2 significantly reduced the messenger RNA level of MsOA2B2 and decreased mortality following amitraz treatment. This study provides both a pharmacological characterization and the gene expression patterns of OA2B2 in M. separata, facilitating further research for insecticides using MsOA2B2 as a target.

BACKGROUND Plutella xylostella has developed resistance to a variety of pesticides in the field. Selection, inheritance, a near-isogenic line, cross-resistance and biochemical mechanisms of pyridalyl resistance were characterized in a field-collected resistant population of P. xylostella from China. RESULTS Compared with a susceptible IVF-S strain, the field-collected FZ population showed ~ 350-fold resistance to pyridalyl. The FZ-PY strain, selected from the FZ population using pyridalyl, developed ~ 640-fold resistance to pyridalyl. Inheritance tests indicated that pyridalyl resistance in the FZ-PY strain was autosomal and incompletely recessive. Through successive backcrossing to IVF-S, a near-isogenic strain (NIL-PY) was established that exhibited 191.21-fold resistance to pyridalyl and no cross-resistance to other tested popular insecticides. No significant effects of synergists and higher activities of metabolic enzymes were observed in NIL-PY compared with IVF-S. Furthermore, the survival rate of NIL-PY larvae, and female oviposition, fecundity and egg viability were markedly reduced in NIL-PY compared with IVF-S. The fitness of NIL-PY was found to be 0.56 compared with IVF-S. CONCLUSION Considering that no relevant effects of synergists or oxidative metabolism were observed in NIL-PY, and that pyridalyl resistance results in significant fitness costs compared with IVF-S, further research will be conducted on the mechanism of target-site resistance. © 2020 Society of Chemical Industry.

Bemisia tabaci, sweetpotato whitefly, is one notorious insect pest on a series of crops worldwide, and many populations show high resistance to various insecticides. The cyantraniliprole-resistant strain of B. tabaci SX-R (138.4-fold) was obtained by selections with an outdoor-collected cyantraniliprole resistant population. By crossing and repeated backcrossing to a susceptible MED-S strain, the trait of cyantraniliprole resistance from SX-R was moved into MED-S to establish one near-isogenic line (CYAN-R). MED-S and CYAN-R were utilized to build patterns of cross-resistance, CYAN-R strain exhibited 63.317-fold resistance to cyantraniliprole, but no cross-resistance to several other successfully commercialized chemical agents. After that significant inhibition of cyantraniliprole resistance by piperonyl butoxide (PBO) and increased cytochrome P450 (3.4-fold) were observed in CYAN-R strain, indicating putative involvement of P450 in detoxification. Furthermore, five published detoxification-related P450 genes in B. tabaci, CYP4C64, CYP6CM1, CYP6CX1, CYP6CX4, and CYP6DZ7 were selected and expression levels of them were measured for exploring mechanisms of cyantraniliprole resistance. Compare with MED-S, no significant overexpression of the five P450 genes was observed in the CYAN-R strain. Above results could be conductive to study on mechanism of cyantraniliprole resistance and will be very helpful for the management of whitefly.

RNAi (RNA interference) is a technology for silencing expression of target genes via sequence-specific double-stranded RNA (dsRNA). Recently, dietary introduction of bacterially expressed dsRNA has shown great potential in the field of pest management. Identification of potential candidate genes for RNAi is the first step in this application. The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae) is a polyphagous, migratory pest, and outbreaks have led to severe crop damage in China. In the present study, two tubulin genes were chosen as target genes because of their crucial role in insect development. Both Msα-tubulin and Msβ-tubulin genes are expressed across all life stages and are highly expressed in the head and epidermis. Feeding of bacterially expressed dsRNA of Msα-tubulin and Msβ-tubulin to third-instar larvae knocked down target mRNAs. A lethal phenotype was observed with knockdown of Msα-tubulin and Msβ-tubulin concurrent with reduction in body weight. Bacterially expressed dsRNA can be used to control M. separata, and tubulin genes could be effective candidate genes for an RNAi-based control strategy of this pest.

In this study, we explored the efficacy of knockdown four genes required for proper nervous system function by RNAi, in the corn earworm Helicoverpa zea (Boddie). Three of these genes encode components of validated insecticide target sites. We synthesized cDNA sequences orthologous to the Drosophila melanogaster genes Para (paralyticts), TipE (temperature-induced paralysis locus E), GluCl (glutamate-gated chloride channel), and Notch, and used these fragments to synthesize double-stranded RNAs (dsRNAs). We then performed experiments in an attempt to induce RNAi-mediated effects on gene expression and viability using three modes of delivery of the dsRNAs: microinjection of eggs, soaking of eggs and feeding of larvae. Microinjection of dsRNAs into eggs induced reduced hatch rates and knockdown of target gene expression for GluCl, para and TipE, but not for Notch. However, neither feeding nor soaking eggs in dsRNA solutions resulted in discernable RNAi effects. These results demonstrated the susceptibility to RNAi effects of the expression of H. zea genes encoding insecticide target sites, which suggests future avenues of research toward practical applications.

The Bemisia tabaci (Gennadius) cryptic species complex comprises important insect pests that cause devastating damage to agricultural crops worldwide. In China, the B. tabaci Mediterranean (MED) (or biotype Q) species is threatening agricultural production all over the country as resistance to commonly used insecticides has increased. This situation highlights the need for alternative pest control measures. Cyantraniliprole, a novel anthranilic diamide insecticide, has been widely employed to control Hemipteran pests. To monitor the levels of resistance to cyantraniliprole in B. tabaci field populations in China, bioassays were conducted for 18 field samples from nine provinces over two years. Compared with median lethal concentration (LC50) for the MED susceptible strain, all field samples had significantly higher resistance to cyantraniliprole. Furthermore, resistance factors (RFs) increased significantly in samples from Shanxi (from 5.62 in 2015 to 25.81 in 2016), Hunan (3.30 in 2015 to 20.97 in 2016) and Hubei (from 9.81 in 2015 to 23.91 in 2016) provinces. This study indicates a considerable decrease in the efficacy of cyantraniliprole against B. tabaci and establishes a baseline of susceptibility that could serve as a reference for future monitoring and management of B. tabaci resistance to cyantraniliprole.

P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.

Objective To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Methods Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood–retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic–reperfusion retinopathy. Results In the OIR model, the study group versus control groups showed a significantly (p

ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in the movement of various substrates, including drugs and insecticides, across the lipid membrane. Demonstration of the role of human ABC transporters in multidrug resistance has led to speculation that they might be an important mechanism controlling the fate of insecticides in insects. However, the role of ABC transporters in insects remains largely unknown. The small brown planthopper, Laodelphax striatellus Fallen, has developed resistance to most of the insecticides used for its control. Our goals were to identify the ABC transporters in La. striatellus and to examine their involvement in resistance mechanisms, using related strains resistant to chlorpyrifos, deltamethrin and imidacloprid, compared with the susceptible strain. Based on the transcriptome of La. striatellus, 40 full-length ABC transporters belonging to the ABCA-ABCH subfamilies were identified. Quantitative PCR revealed that over 20% of genes were significantly up-regulated in different resistant strains, and eight genes from the ABCB/C/D/G subfamilies were up-regulated in all three resistant strains, compared with the susceptible strain. Furthermore, synergism studies showed verapamil significantly enhanced insecticide toxicity in various resistant strains but not in the susceptible strain. These results suggest that ABC transporters might be involved in resistance to multiple insecticides in La. striatellus.

The Bemisia tabaci (Gennadius) cryptic species complex comprises very destructive insect pests for agricultural crops worldwide. In China, the B. tabaci MED species (also known as biotype ‘Q’), has supplanted the MEAM1 species (biotype ‘B’) which is threatening agricultural production around the country. The new anthranilic diamide insecticide, cyantraniliprole, provides one novel step for the management of B. tabaci and the development of resistance to other insecticides. Ryanodine receptors of insect are the main target sites of the diamide insecticides. In this study, the full-length cDNA of a ryanodine receptor gene ( BtRyR ) was cloned and characterized from B. tabaci MED. The cDNAs of BtRyR contain a 15,369-bp open reading frame with encoding 5122 amino acids (GenBank ID: KY244091). BtRyR shares 76–83% identity with other insect RyR isoforms and 42–45% identity with vertebrate RyR isoforms. Spatial and temporal expression of BtRyR mRNA was at the highest relative level in pseudopupae and head, and at the lowest expression level in egg and abdomen. The expression levels of whole body BtRyR mRNA were increased remarkably after insecticide-treatments of adults with cyantraniliprole at 0.01 to 1 mg/l. This structural and expression data on BtRyR provides the basis for further understanding the selective action of cyantraniliprole.

DNA methylation refers to the addition of cytosine residues in a CpG context (5'-cytosine-phosphate-guanine-3'). As one of the most common mechanisms of epigenetic modification, it plays a crucial role in regulating gene expression and in a diverse range of biological processes across all multicellular organisms. The relationship between temperature and DNA methylation and how it acts on the adaptability of migratory insects remain unknown. In the present work, a 5,496 bp full-length complementary DNA encoding 1,436 amino acids (named MsDnmt1) was cloned from the devastating migratory pest oriental armyworm, Mythimna separata Walker. The protein shares 36.8-84.4% identity with other insect Dnmt1 isoforms. Spatial and temporal expression analysis revealed that MsDnmt1 was highly expressed in adult stages and head tissue. The changing temperature decreased the expression of MsDnmt1 in both high and low temperature condition. Besides, we found that M. separata exhibited the shortest duration time from the last instar to pupae under 36°C environment when injected with DNA methylation inhibitor. Therefore, our data highlight a potential role for DNA methylation in thermal resistance, which help us to understand the biological role adaptability and colonization of migratory pest in various environments.

As well as arising from single point mutations in binding sites or detoxifying enzymes, it is likely that insecticide resistance mechanisms are frequently controlled by multiple genetic factors, resulting in resistance being inherited as a quantitative trait. However, empirical evidence for this is still rare. Here we analyse the causes of up-regulation of CYP6FU1, a monoxygenase implicated in resistance to deltamethrin in the rice pest Laodelphax striatellus. The 5′-flanking region of this gene was cloned and sequenced from individuals of a susceptible and a resistant strain. A luminescent reporter assay was used to evaluate different 5′-flanking regions and their fragments for promoter activity. Mutations enhancing promoter activity in various fragments were characterized, singly and in combination, by site mutation recovery. Nucleotide diversity in flanking sequences was greatly reduced in deltamethrin-resistant insects compared to susceptible ones. Phylogenetic sequence analysis found that CYP6FU1 had five different types of 5′-flanking region. All five types were present in a susceptible strain but only a single type showing the highest promoter activity was present in a resistant strain. Four cis-acting elements were identified whose influence on up-regulation was much more pronounced in combination than when present singly. Of these, two were new transcription factor (TF) binding sites produced by mutations, another one was also a new TF binding site alternated from an existing one, and the fourth was a unique transcription start site. These results demonstrate that multiple cis-acting elements are involved in up-regulating CYP6FU1 to generate a resistance phenotype.

RNA interference (RNAi) efficiency dramatically varies among different insects and among administration methods. Numerous studies have revealed that a poor RNAi response is usually associated with a high double-stranded RNA (dsRNA)-degrading activity. Using the red flour beetle Tribolium castaneum, we conducted genome-wide identification of genes encoding dsRNA-degrading nucleases of the DNA/RNA non-specific endonuclease superfamily. To achieve a robust RNAi response in T. castaneum, four dsRNase genes were identified in the genome that seemed to be the potential factors reducing RNAi efficacy. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH 8.0) in the absence of Mg2+ at 37 °C. The dsRNA-degrading activity was predominantly present in the gut, and via heterologous expression and RNAi experimentation, gut-specific TcdsRNase1 was confirmed as the major nuclease performing dsRNA degradation. After a knockdown of the TcdsRNase1 nuclease activity, RNAi efficiency improved from 38.6% to 58.9% and from 20.9% to 53.9% for injection and ingestion of dsRNA, respectively. Our results contribute to a comprehensive understanding of the mechanisms influencing dsRNA stability and even RNAi efficiency in T. castaneum and point to a good method for improving RNAi efficiency through downregulation of the relevant nuclease activity.

Bemisia tabaci (Gennadius) is one of the world's most devastating crop pests, and many populations show high resistance to various insecticides. To determine B. tabaci susceptibility to flupyradifurone and understand mechanisms of resistance, we conducted field monitoring and cross-resistance, synergism, and metabolic enzyme activity assays with two resistant strains: Wuhan (WH) and Xiangyang (XY). Results showed that most field populations were highly susceptible to flupyradifurone. However, WH and XY populations, with resistance ratios of 21.95- and 29.03-fold, respectively, showed a low level of flupyradifurone resistance but no cross-resistance to imidacloprid or cyantraniliprole. Synergism tests demonstrated that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) significantly inhibited flupyradifurone resistance in the XY strain, while PBO significantly inhibited resistance in the WH strain. Increased cytochrome P450 monooxygenase and esterase activity correlated with flupyradifurone resistance in the two resistant strains, indicating putative involvement of these enzymes in detoxification. By rotating flupyradifurone with other chemical agents that lack cross-resistance, development of resistance could be delayed, making flupyradifurone an effective agent for whitefly management in China.

The Bemisia tabaci (Gennadius) cryptic species complex includes important crop pests, and among them, the cryptic species Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) cause substantial crop losses in China. The second-generation neonicotinoid clothianidin acts as an agonist of the nicotinic acetylcholine receptor in the insect nervous system and has both stomach and contact activity. In this study, the toxicity of clothianidin and five other insecticides to MED and MEAM1 was examined. The sublethal effects of clothianidin on the development and reproduction of MED and MEAM1 were also investigated. Among the six insecticides tested, clothianidin showed toxicities to both MED and MEAM1 adults with LC50 values of 5.23 and 5.18 mg/liter, respectively. The sublethal effects of clothianidin were assessed by treating MED and MEAM1 adults with the LC25 of 1.58 and 1.13 mg/liter, respectively. The LC25 treatments accelerated the development of the F1 generation but reduced survival and fecundity of both species. Our results indicate that clothianidin could be useful for the management of B. tabaci MED and MEAM1.

The ryanodine receptors of insects are the main target sites of diamide insecticides, which show highly selective insecticidal activity relative to toxicity in mammals and provide a novel option for managing lepidopteran pests. The oriental armyworm, Mythimna separata (Walker), is a destructive pest of agricultural crops, and great efforts have been undertaken to control this pest including repeated insecticide applications. In this study, full-length cDNA of a ryanodine receptor gene from M. separata (MsRyR) was cloned and characterized. The cDNA of MsRyR had a 15,372 bp open reading frame and encoded 5124 amino acids (GenBank ID: MG712298). MsRyR shares 78-97% identity with RyR isoforms of other insects, and50% identities with Homo sapiens RyRs 1-3. Temporal and spatial expression analysis detected MsRyR at all developmental stages and in all tissues. The highest relative levels of MsRyR were detected in the second instar and head. Exposure to chlorantraniliprole after 24 h significantly increased the expression levels of whole body MsRyR mRNA. In addition, dietary ingestion of dsMsRyR significantly reduced the mRNA level of MsRyR and greatly decreased chlorantraniliprole-induced mortality. Our results revealed that the MsRyR could be the molecular target of chlorantraniliprole, and provided the basis for further understanding the resistance mechanism of chlorantraniliprole.

RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate the induction of RNAi in the codling moth, Cydia pomonella , a major lepidopteran pest of apple, pear, and walnut. Prior to a knockdown screen, fluorescently labeled small interfering RNA (siRNA) and double-stranded RNA (dsRNA) derived from green fluorescent protein (GFP) coding sequence were delivered to the surface of artificial diet to which neonate larvae were introduced and subsequently examined for the distribution of fluorescence in their tissues. Fluorescence was highly concentrated in the midgut but its presence in other tissues was equivocal. Next, dsRNAs were made for C . pomonella genes orthologous to those that have well defined deleterious phenotypes in Drosophila melanogaster . A screen was conducted using dsRNAs encoding cullin - 1 ( Cpcul1 ), maleless ( Cpmle ), musashi ( Cpmsi ), a homeobox gene ( CpHbx ), and pumilio ( Cppum ). The dsRNAs designed from these target genes were administered to neonate larvae by delivery to the surface of the growth medium. None of the dsRNA treatments affected larval viability, however Cpcul1 -dsRNA had a significant effect on larval growth, with the average length of larvae about 3 mm, compared to about 4 mm in the control groups. Measurement of Cpcul1 transcript levels by quantitative real-time PCR (qRT-PCR) revealed a dose-dependent RNAi effect in response to increasing amount of Cpcul1 -dsRNA. Despite their reduced size, Cpcul1 -dsRNA-treated larvae molted normally and matured to adulthood in a manner similar to controls. In an additional experiment, Cpcul1 -siRNA was found to induce similar stunting effect as that induced by Cpcul1 -dsRNA.

The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Erebidae), is an important pest in the Australian pine tree, Casuarina equisetifolia, forest in the coastal area of South China. At the same time, as a closely related species of Lymantria dispar L. (Lepidoptera: Erebidae), it is also a potential quarantine pest. In the present study, specific primers were designed for identification of L. xylina based on the COI barcoding sequence between L. xylina and four other common forest pests. A 569-bp fragment was successfully amplified from 40 L. xylina from five geographical populations in four Chinese provinces. In addition, even through the analysis came from five highly diverse populations of L. xylina, the genetic distances ranged from 0.001 to 0.031. The neighbor-joining tree showed that the species from Hubei and Chongqing were clustered within a distinct group.

The Bemisia tabaci (Gennadius) cryptic species complex comprises very destructive insect pests of agricultural crops worldwide and has been found to be resistant to various insecticides in China. Abamectin is one of the most widely used insecticides for insect pest control and the glutamate-gated chloride channel (GluCl) in insects was presumed to be the main target site of abamectin. In this study, a 1353 bp full-length cDNA encoding GluCl (named BtGluCl, GenBank ID: MF673854) was cloned and characterized from B. tabaci. BtGluCl encodes 450 amino acids, which shares 71–81% identity with other insect GluCl isoforms. Spatial and temporal expression revealed BtGluCl was highly expressed in the 4th nymphal instar and adult head, and the least expressed in the 1st nymphal instar and adult leg. Dietary ingestion of dsBtGluCl significantly reduced the mRNA level of BtGluCl in the treated adults by 62.9% and greatly decreased abamectin-induced mortality. Thus, our results could be conducive to further understanding the mechanisms of resistance to abamectin in arthropods.

The Chilo infuscatellus (Snellen; Lepidoptera: Pyralidae) is the main pest of sugarcane in China. The shortage of easily distinguishable morphological characters especially in early stage make it challenging to diagnosed and promptly take steps for pest management. In the present study, we described a PCR method for the molecular identification of based on barcode region of COI sequences between C. infuscatellus and four other sugarcane borer species. A 285bp fragment was successfully amplified from all life stages and different geographical populations. Sensitivity tests revealed that diagnostic bands were generated as low as the DNA template concentration of 5 ng/µl. Our work demonstrated a rapid and accurate way for the molecular diagnosis of C. infuscatellus.

Background Even though both dsRNA and siRNA have been widely used in laboratory studies to knock down target genes, it has been found that siRNA rarely triggers phenotypic changes for intact insects. In this study, Tribolium castaneum was used as a model species to compare dsRNA and siRNA and select the more efficient of these for insect RNAi. Results Four dsRNAs and ten siRNAs targeting the same three genes were tested by checking the suppression of target genes and the deformation of treated insects. The results showed that dsRNAs could reduce the expression of target genes by 55–75% for more than 7 days and deformed 78.8–87.0% of the larvae tested. Injection of their corresponding siRNAs targeting the same gene did yield about 45% silencing of target genes, but only in a short time period (from day 2 to day 4), and it did not trigger any phenotypic changes. Conclusion SiRNAs were less efficient than their corresponding dsRNAs. Most of them could hardly trigger any phenotypic changes owing to the temporary silencing effect on target genes. Meanwhile, the efficiency of different siRNAs varied with their matching sequence, and careful selection was necessary for proper use. © 2013 Society of Chemical Industry

Introduction. Bone marrow-derived mesenchymal stromal cells (BMSCs) have emerged as promising cell candidates but with poor survival after transplantation. This study was designed to investigate the efficacy of VEGF, bFGF, and IGF-1 on BMSCs’ viability and proliferation bothin vivoandin vitrousing bioluminescence imaging (BLI).Methods. BMSCs were isolated fromβ-actin-Fluc+transgenic FVB mice, which constitutively express firefly luciferase. Apoptosis was induced by hypoxia preconditioning for up to 24 h followed by flow cytometry and TUNEL assay. 106BMSCs with/without growth factors were injected subcutaneously into wild type FVB mice’s backs. Survival of BMSCs was longitudinally monitored using bioluminescence imaging (BLI) for 5 weeks. Protein expression of Akt, p-Akt, PARP, and caspase-3 was detected by Western blot.Results. Hypoxia-induced apoptosis was significantly attenuated by bFGF and IGF-1 compared with VEGF and control groupin vitro(P<0.05). When combined with matrigel, IGF-1 showed the most beneficial effects in protecting BMSCs from apoptosisin vivo.The phosphorylation of Akt had a higher ratio in the cells from IGF-1 group.Conclusion. IGF-1 could protect BMSCs from hypoxia-induced apoptosis through activation of p-Akt/Akt pathway.

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

We have investigated the contributions of three dominant macrophyte species Deyeuxia angustifolia Carex lasiocarpa and Carex pseudocuraica (covering about 10 304 km(2)) to carbon (C) nitrogen (N) and phosphorus (P) stocks in the largest freshwater marsh (17 300 km(2)) in China for a 3 year period (from 2002 to 2004) The monthly biomass seasonal and annual net primary productivity (NPP) and nutrient concentrations of three species were measured All three plant species showed rapid growth in the rainy season The maximum and minimum production rates in the freshwater marsh were similar to 36 19 and similar to 9 92 g m(-2) day(-1) respectively The total NPP accounts 1900-2700 g m(-2) year(-1) in the studied area Total organic carbon (TOC) total nitrogen (TN) and total phosphorus (IT) concentrations in roots were higher than those in stem and leaf tissues The vast beds of the three studied species comprise 80% of the grass covered marsh of Sanjiang plain contributing annual nutrient stocks of similar to 10 99 x 10(6) similar to 788 36 x 10(3) and similar to 18 10 x 10(3) t (tonnes) for TOC TN and IT respectively Our results suggest that the nutrient bioaccumulation capacity in freshwater marshes depend mainly on plant species which are decided by hydrological conditions The nutrient stocks in the Sanjiang plain marsh have been greatly reduced because some of the area occupied by C lasiocarpa was replaced by D angustifolia as a result of succession caused by the changes of water table

The west Jilin Province is a typical area in the ecotone between agriculture and animal husbandry, with a frangible eco-environment. With respect to the three aspects of water resource, natural disasters and land degradation, 10 indices were selected to establish a matter-element model for the assessment of eco-environmental frangibility in the west Jilin Province. The results indicate that during 1985-2000, Qian’an, Fuyu, and Changling had the least frangibility (level I), followed by Da’an and Qianguo (level II), and Taobei, Zhenlai, Taonan, and Tongyu had the highest frangibility (level III). On the whole, the counties in Songyuan city were less frangible than those in Baicheng city. Different counties had different frangibilities to environmental factors, e.g., Da’an and Tongyu were frangible in water resource conditions; Taobei, Zhenlai, Taonan, Tongyu and Qian’an suffered most from natural disasters; while Taobei, Taonan and Qianguo were threatened by severe land degradation.

Plant growth, biomass allocation and root distribution were investigated in the submerged macrophyte Vallisneria natans growing in heterogeneous sediments. Experimentally heterogeneous sediment environments were constructed by randomly placing 4 cm of clay or sandy loam into the top (0-4 cm) or bottom (4-8 cm) layer within an experimental tray, providing two homogeneous and two heterogeneous treatments. Biomass accumulation was significantly affected by the experimental treatments: higher in the homogeneous sediment of clay (32 mg per plant) and the two heterogeneous treatments (about 27 mg per plant), but lower in the homogeneous sediment of sandy loam (15 mg per plant). Root: shoot ratio was also different among the four treatments. Compared with the treatments of clay in the top layer, plants allocated more biomass to roots at the treatments of sandy loam in the top layer. Heterogeneous sediments significantly affected root distribution pattern. Compared with the treatments of sandy loam in the bottom layer, root number (7-8 versus 13-14) and total root length (3.6-4.0 cm versus 29.5-40.0 cm) in the bottom layer were significantly higher in the treatments with clay in the bottom layer. These results indicate that both sediment structure and nutrient availability influence growth and root system distribution of V natans. (c) 2006 Elsevier B.V. All rights reserved.

Red-crowned Crane (Grus japonensis), a kind of endangered large-scale wader, is one of valuable and rare birds in the world. It has been indexed in China Red Data Book of Endangered Animals (Aves), China Data Book of Major Protection Wild Animals, World Red Data Book of Birds, State Trade Protocol of Endangered Wild Plants and Animals Species. The paper studies the amount, distribution, habit, growth and habitat of Red-crowned Crane in China in detail, systematically analyses the ecological environment feature of Red-crowned Crane breeding area, including the vegetation feature, the vertical distribution law of absolute humidity and temperature, some carnivorous food and herbivorous food for Red-crowned Crane. At present, the ecological environment of breeding area has been destroyed seriously by human activity. The area of marshland is reduced rapidly. A large area of reeds has been disappeared. The survival of Red-crowned Crane is threatened greatly. So it’s necessary to maintain the biodiversity of breeding area in order to protect Red-crowned Crane.

In this paper, an incubation test with soil columns was carried out for 35 days to study the dynamics of DOC leaching from the organic layers (2 approximately 13 cm) of two meadow marsh soils, and the correlation of DOC release with soil respiration, NH4+ formation, and leachate pH. The results showed that in the first week, DOC release presented a fast decrease, and then held on a steady level, which could be well described by one order exponential equation (R2 > 0.96, P < 0.05). During 35 days incubation, the cumulative release of DOC from two test soils was 210.9 microg C x g(-1) and 506.58 microg C x g(-1), and the cumulative emission of CO2 was 679.64 microg C x g(-1) and 455.54 microg C x g(-1), respectively. It was suggested that in the course of incubation, the lower DOC release might be related to the microbial carbon limitation because of the greater CO2 production rate. A significantly positive correlation was found between DOC release and NH4+ formation (rXI = 0.886, P