With 13C glucose as the trophic link, Pyro-SIP revealed that a relatively simple but distinctive gut microbiota co-developed with the host, both metabolic activity and composition shifting throughout larval stages. Pantoea, Citrobacter and Clostridium were particularly active in early-instar, likely the core functional populations linked to nutritional upgrading. Enterococcus was the single predominant genus in the community, and it was essentially stable and metabolically active in the larval lifespan. Based on that Enterococci formed biofilm-like layers on the gut epithelium and that the isolated strains showed antimicrobial properties, Enterococcus may be able to establish a colonization resistance effect in the gut against potentially harmful microbes from outside. Not only does this establish the first in-depth inventory of the gut microbiota of a model organism from the mostly phytophagous Lepidoptera, but this pilot study shows that Pyro-SIP can rapidly gain insight into the gut microbiota's metabolic activity with high resolution and high precision.The gut microbiota is of crucial importance for the host with considerable metabolic activity. Although great efforts have been made toward characterizing microbial diversity, measuring components' metabolic activity surprisingly hasn't kept pace. Here we combined pyrosequencing of amplified 16S rRNA genes with  Not only thriving in the gut, these inner microbial residents also contribute to host metabolism through nutrient release, xenobiotic detoxification and immune regulation, all of which greatly boost host fitness 13C)-labeled carbon source into growing microbes and the selective recovery of the isotope-enriched cellular components, such as the most informative nucleic acids, which could provide specific taxonomic information. 13C-enriched “heavy” DNA or RNA can be separated from unlabeled, normal “light” (12C) nucleic acids by density-gradient ultracentrifugation and subsequently retrieved from gradient fractionation procedure for further molecular analysis et al. introduced a combination of DNA-SIP and pyrosequencing, namely Pyro-SIP, in SIP gradient interpretation Stable isotope probing (SIP) is a promising, culture-free technique that is often used in environmental microbiology to identify active microorganisms involved in various biogeochemical processes Spodoptera littoralis , a highly polyphagous pest found worldwide that causes considerable yield losses of many economically important crops. The devastating larval stage of S. littoralis (cotton leafworm) consumes large amounts of plant material in a short time, liberating abundant plant-derived saccharides mainly glucose in the alimentary canal to be a most important carbon and energy source for both host and active gut bacteria. More recently, the microbiology of this important insect has received increasing attention, a complex and variable commensal microbiota is found in the larval gut As a first attempt, we successfully conducted this refined Pyro-SIP to unravel the metabolically active bacterial community in the gut of an insect model organism, 13C glucose as the trophic link, Pyro-SIP revealed that a relatively simple but distinctive gut microbiota co-develops with the host, both metabolic activity and composition shifting throughout larval stages. Three families, including Enterococcaceae, Clostridiaceae and Enterobacteriaceae, are particularly rich and active inside, which likely represent the core functional populations in the gut linked to nutritional upgrading and pathogen defense for improving larval fitness. This study also establishes the first in-depth inventory of the gut microbiota of a model organism from mostly phytophagous Lepidoptera. Knowledge of the gut bacteria of such a major insect herbivore could lead to new targets for pest control.Using Sugar composition was analyzed in different gut regions, namely the foregut, midgut and hindgut. Gut contents collected from larvae fed on their native diet (cotton) or defined artificial diet (without antibiotics or preservatives) were analyzed by gas chromatography-mass spectrometry (GC-MS) after water extraction and aldononitrile acetate derivatization, which is a rapid and sensitive derivatization method for non-volatile saccharides and each sugar forms a single chromatographic peak after GC separation The GC-MS profile indicated that several pentose and hexose sugars were present in the gut, including ribose, arabinose, mannose, glucose and galactose . HoweverAs the major sugar, glucose was quantified based on a specific enzymatic assay, which revealed that maximal glucose concentrations occurred in the foregut and then dropped from the anterior to posterior end of the gut. In cotton-feeding larvae, the average concentration of glucose in the aqueous phase of the foregut was approximately 17.7 mM, significantly higher than that in the hindgut, which was around 9.8 mM . The rapin vivo SIP by performing 13C-glucose amendment. Initial concentrations of glucose in the foregut of larvae fed on cotton reached 20 mM, but only about 10 mM in larvae fed on artificial diet  was used in the control group. We tested the labeling process by feeding early-instar larvae (from 1st to 2nd) on glucose-amended artificial diets for 24 and 48 hours, respectively. For revealing the difference during larval development, the same process was also employed in the study of late-instar larvae (5th).As the most effective energy and carbon source available, nutritive glucose in the gut provided a good chance to carry out ial diet . Thus, t13C treatment and unlabeled control group were dissected at each time point, and total DNA was extracted from fresh gut tissues. Isotopic ratio mass spectrometry (IRMS) measurements of δ13C in the extracted DNA showed substantial isotope enrichment in both 24 h (δ13C = 33.6±5.3‰) and 48 h (δ13C = 141.2±33.1‰) labeling samples, compared to natural stable isotope abundance in the control (δ13C = −30.7±1.3‰)  . Feeding−1 to 1.761 g ml−1, which was in the expected range according to previous reports Both the labeled and control samples were conducted in the same batch of isopycnic ultracentrifugation to minimize potential variation during this process. After 40 h ultracentrifugation, formed gradients were fractionated into 12 equal aliquots, and the abundance of DNA present in each individual fraction was quantified, allowing us to compare DNA template distribution in the formed gradients . The den13C treatment, which had a new peak of abundant DNA at the buoyant density (BD) of 1.730 g ml−1 (fraction 5). After 48 h, the peak shifted to an even heavier fraction at BD of 1.735 g ml−1 (fraction 4), indicating more 13C incorporation into DNA, which confirmed the IRMS data. By contrast, most of DNA from the unlabeled control was still distributed over light fractions (BD was <1.718 g ml−1) and no peak shifted towards the high BD  in order to fingerprint the community. Changes became visible when the DGGE profile derived from the 13C treatment was compared with that from the unlabeled control. Several strong bands were clearly detected from heavy fractions (3–6) of the labeled sample, whereas almost no pattern changed over all gradients of the control , which was caused by the increased amount of  control . Those u−1 into a compiled “heavy” fraction. BDs ranged around 1.718 g ml−1 in a “middle” fraction and from 1.688 to 1.705 g ml−1 in a “light” fraction for subsequent pyrosequencing.Based on the fingerprinting analysis, we combined fractions with BDs ranging from 1.730 to 1.735 g mlQuantitative pyrosequencing was performed directly on representative SIP gradients to reveal species lineage and relative abundance. PCR amplification using archaea- or fungus-specific primers failed to amplify any archaeal 16S rRNA or fungal ITS sequences. Cultivation-based attempts to recover fungi from the gut with three general fungus-growing agar plates were also unsuccessful .Bacteria-specific primer for pyrosequencing amplified the V1–V3 region of 16S rRNA gene for taxonomic classification. After denoising and removing chimeric sequences, 120,045 high quality reads were generated, with an average length of 404 nucleotides. There was a low incidence of sequences unclassified at the phylum level (maximum 3.82%). For all samples, the rarefaction curve tended towards saturation at similar numbers of sequences at the species level, indicating the sampling was comprehensive  and 4A. Comamonas and Stella, had more chance to be amplified and showed an increased proportion in the profile of [12C]-Heavy fraction , Micrococcus and Propionibacterium (Actinobacteria) appeared in a small number of sequences.However, in the context of Proteobacteria, late-instar larvae mainly reduced the proportion of Gammaproteobacteria. S. littoralis , while negligible portions of these sequences were detected from the heavy fraction of the control  revealed a large amount of Enterococci closely adhered to the mucosal layer of gut epithelium, where they formed a thick biofilm-like structure  and Clostridium apparently were more active in early-instar larvae, while Enterococcus became strongly active in fully grown late-instar larvae  is found. Not only do they consistently occur throughout the whole larval lifespan, these bacteria are also persistently identified from different sampling batches of normal larvae, indicating they are the indigenous gut residents and may be the true symbionts of S. littoralis.Comprehensive analysis of the recovered DNA revealed a relatively simple but distinctive gut microbiota that co-develops with the host; both the composition and metabolic activity sustainably shift throughout larval stages. Overall, pyrosequencing reads were dominated by taxa from Proteobacteria and Firmicutes. A similar pattern has recently been described in fruitfly larvae Pantoea and Citrobacter from the family Enterobacteriaceae, was the most active group. Bacteria in this phylum closely associate with insect herbivores and possess broad polysaccharide-degrading abilities Enterobacteriaceae isolated from silkworm (Bombyx mori) gut had the ability to secrete enzymes important in the digestion of complex dietary plant biopolymers such as cellulose, xylan, and pectin Pantoea strain from a woodwasp (Sirex noctilio) detected genes putatively encoding for carbohydrate-active enzymes, with the majority predicted to be active on hemicellulose and more simple sugars in vitro growth assays and genomic investigation provide an initial picture of potential metabolic capabilities but do not offer information on bacterial activity in situ. Our Pyro-SIP results are the direct evidence of microbial metabolic activity in vivo, supporting the previous hypothesis that Gammaproteobacterial symbionts are involved in carbohydrate degradation. In spite of its low titer, Clostridium was another highly active group in early-instar larvae; these obligate anaerobes are capable of forming endospores. The colonization of Clostridia has commonly been linked to its highly efficient cellulose digestion and its ability to ferment a variety of sugars Clostridia may enhance host immunity Enterobacteriaceae and Clostridiaceae synergistically participate in the digestive process in the gut, facilitating the breakdown of organic substrates in the foliage and making them more suitable for the host's digestion, absorption and metabolism. Given that sufficient nutrient supply is important in the early life, the released simple sugars would directly benefit early larval development, which further could be fermented to various other nutrients such as short-chain fat acids, greatly promoting the gut ecological environment. These bacteria, widely distributed in nature, could be easily acquired when larvae forage. The large sampling depth of this study revealed a number of rare taxa in the gut. Although this variety diminished over time, it is possible that rare members perform some of the microbiota's functions or have a role in special situations, as suggested by previous studies In early-instar, a diverse assembly of Proteobacteria, especially Gammaproteobacteria dominated by Clostridium proliferation. Enterococcus predominated and was detected as the most active group. The successful expansion of Firmicutes over time probably in turn suppressed the growth of bacteria from other phyla in the same habitat, particularly Proteobacteria. Linked with larval development, changes in insect physiology such as dropped redox potential and enhanced host immunological response could be potential driving factors for observed changes. As larvae grow bigger, less oxygen can penetrate into the gut lumen over the thicker gut wall and elongated alimentary tract, which cause consistently low oxygen tension in the gut compartment Clostridium and facultative anaerobic Enterococcus.In late-instar, the gut microbiota became even simpler with a large decrease of microbial diversity and richness. More than 97% of community members belonged to Firmicutes, partially because of a significant Enterococcus, a Gram-positive lactic acid bacterium (LAB), is the single predominant genus in the community. LAB are well-recognized beneficial organisms of the gut microbiota of many animals, including insects Enterococci are essentially stable throughout Spodoptera's lifespan and are vertically transmitted from mother to offspring via egg, as shown for ManducaEnterococcus likely controls the whole microbiota together with the host, which may be crucial for establishing a distinctive and reoccurring gut community. Besides in cotton leafworm, Enterococcus is present in diverse Lepidoptera, such as the larva of gypsy moth (Lymantria dispar) and cotton bollworm (Helicoverpa armigera) from both field and laboratory-reared samples Enterococcus was found to be already active on the eggs of tobacco hornworm (Manduca sexta) based on rRNA detection Enterococcus may have important functional implications for Lepidoptera in general. FISH indicated that a high number of Enterococci closely attach to the mucus layer of gut epithelium to form a biofilm-like structure, which may be the reason for its high stability in the gut of healthy larvae and, furthermore, may prevent the gut from being invaded by harmful pathogenic microbes Enterococcus is likely a defensive symbiont for the health of the host. Considering cotton leafworm is a generalist feeder in the field, its digestive tract is constantly challenged by the potentially harmful bacteria and fungal endophytes that it ingests. Enterococci, maintained in biofilm-like structure and showing potent antimicrobial properties, may be able to establish a colonization resistance effect in the gut, which protects the host against pathogens and a wide range of noncommensal competing microbes from outside Enterococci do not need fully turn on their metabolic machinery in a semisterile laboratory rearing environment, which may explain their low metabolic activity in early-instar. Not only are they related to nutritional upgrading and pathogen defense, active bacteria may also contribute to other gut metabolism, such as detoxifying plant-derived noxious allelochemicals and regulating the host's immune homeostasis. Metagenomic analysis of the labeled DNA from this study will increase our knowledge of other functions supplied by the active microbiota.In vivo activity measurement offered by SIP helps to better understand the change and maintenance of the microbial community and gives further insights into the role of active members in host fitness. Our data also established the first in-depth inventory of the gut microbiota of a model organism from mostly phytophagous Lepidoptera. Knowledge of the gut bacteria in such a major herbivore insect may also provide new targets for agricultural pest control.During coevolution, indigenous gut bacteria have adapted to work together in this distinct ecological niche and supply their metabolic benefits to the host. On the other hand, different physicochemical conditions with respect to host development, such as gut alkalinity, oxygen tension, might impact the community's activity, which in turn influences its composition and consequently metabolic functions. In that manner, both the host and true symbionts elaborately regulate their own metabolic activity and efficiency to preserve the host-microbial mutualism. This pilot study shows Pyro-SIP could rapidly gain insight into not only the structure of the community but, more important, its components' local metabolic activity with high resolution and high precision, which provides a starting point for research on more complex ecosystems, such as termite or human microbiota. Although our work has focused on the discrimination of general active gut bacteria by tracking the ubiquitous glucose, similar analysis can be performed on more specific carbon sources, for instance, labeling plant defense compounds to assess active bacteria involved in the host detoxification process. Therefore, Pyro-SIP also provides another way to understand the complex gut metabolism by breaking it down. With the development of such new approaches, a revolution in understanding of the inner world of life may come into reach soon.Spodoptera littoralis  were hatched and reared on a sterile artificial diet, made of white bean and essential nutrients without addition of antibiotics or preservatives. All cotton plants (Gossypium hirsutum DP90) were cultivated in our greenhouse under standard conditions . To analyze the sugar composition in the gut, larvae were grown on cotton seedlings in pots or on fresh artificial diet in boxes, respectively. After 7 days of feeding, larvae were washed, sedated on ice and dissected to collect gut contents under a dissecting microscope. The alimentary canal was divided into three regions  with sterile scissors (st to 2nd) or late-instar larvae (5th) for 24 and 48 h using glucose-amended artificial diet. The artificial diet was frequently changed to avoid any contamination. The whole guts of larvae were collected as above for DNA extraction. For each analysis, the experiment was performed in triplicate.scissors , and gut−1. The temperature program was set as follows: 80°C (2 min), then at 15°C min−1 to 300°C (6 min). Data were acquired and processed using the software Xcalibur . Mass spectra were taken in the selected ion monitoring (SIM) mode at 70 eV. All compounds were identified by comparing their retention times and MS data with authentic references. An aliquot of the extract was also hydrolyzed with 2 M trifluoroacetic acid  at 100°C for 2 h, solubilizing the matrix polysaccharides into their monosaccharides, and subsequently derivatized to corresponding aldononitrile-acetates. Further GC-MS analysis was identical to those of non-hydrolyzed extracts. Glucose concentration was verified using a glucose assay kit based on the specific and sensitive enzymatic method according to the protocol supplied by the manufacturer .For the characterization of soluble organic compounds in the alimentary canal, freshly collected gut content was extracted according to the published literature All freshly collected gut samples were dried at 45°C in a Speedvac  and crushed in a 1.5 ml Eppendorf tube with a sterile plastic pestle. Genomic DNA was extracted using the PowerSoil™ DNA Isolation Kit  according to the manufacturer's protocol. An additional heating step at 65°C for 10 min was included just prior to bead-beating. After purification, DNA concentrations were quantified with the NanoVue spectrophotometer . The successful extraction of microbial metagenomic DNA from the gut was verified by using PCR assays with general bacterial 16S rRNA primers (27f and 1492r) 13C enrichment in extracted nucleic acids. The 13C composition was determined by a coupled elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Purified DNA samples were placed into 0.04 ml tin capsules  and dried overnight. Samples were completely converted to CO2, N2 and H2O after combustion  in a constant helium flow (80 ml min−1) by using an Elemental Analyzer . After passing a water trap (MgClO4), the gases were separated chromatographically at 85°C and transferred via the split valve to a coupled isotope ratio mass spectrometer (IRMS) . The acetanilide standard was calibrated against the international standard Vienna Pee Dee Belemnite (VPDB) using NBS 22  with a δ13C value of −30.03‰ Carbon isotopic ratio can be a reliable indicator of the −1 using a published protocol 13C-labeled DNA was separated by isopycnic ultracentrifugation under 50,000 r.p.m. in a NVT90 rotor in an Optima L-90K ultracentrifuge (Beckmann) at 20°C for 40 h with vacuum and subsequently the formed density gradients were fractionated from bottom to top into 12 equal fractions (425 µl each) by displacement from above with water using a HPLC pump at a flow rate of 850 µl min−1   solution with an average density of 1.725 g mlGermany) . FractioSeparated gradient fractions were screened for differences in bacterial community composition by denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial 16S rRNA gene was amplified from all fractions in two steps as a nested PCR with the primer set 27f/1492r and 968F/1401R . DGGE wat-test against the taxonomic data extracted from QIIME. The significance level to threshold  was set at 0.05 Representative gradients were submitted to pyrosequencing as described previously Enterococcus-specific probe was used for detection and images were taken with an Axio Imager Z1 microscope  To localize the dominant gut symbionts, FISH was performed on 5 µm thin cross sections of the cold polymerizing resin  embedded gut tissue. The specificity of probes was tested and hybridization condition was achieved as described Enterococci were isolated from cotton leafworm gut by Enterococcus-selective agar plate  and 16S rRNA gene was amplified and sequenced. The antimicrobial activity of isolated strains against different indicator bacteria was evaluated using an agar diffusion assay. The overnight culture supernatant of an E. mundtii strain in MRS broth  at 30°C was adjusted to pH 7.0 and filtered through 0.22 µm PVDF membrane. Holes  were cut out from the BHI agar plate (Roth) inoculated with indicator strains (Micrococcus luteus or Leuconostoc mesenteroides) and filled with 60 µl of E. mundtii culture filtrates. The agar plates were incubated at 30°C for 24 h, and antimicrobial activity was detected by the formation of clearance zones around loading holes.The pyrosequencing data have been deposited at the NCBI GenBank Short Read Archive under accession no. SRA057979.Table S1Primers and probes used for the characterization and localization of bacterial taxa in the gut of Spodoptera littoralis larva.(DOCX)Click here for additional data file.Figure S1Sugar composition of the gut content after acid hydrolysis. Larvae fed on cotton plants.(TIF)Click here for additional data file.Figure S2Sugar composition of the gut content after acid hydrolysis. Larvae fed on artificial diet.(TIF)Click here for additional data file.Figure S313C-glucose labeling and the native glucose control (12C-glucose).The DGGE fingerprinting of density-revealed gradient fractions from the  (a) Fraction-dependent PCR assay of amplifying bacterial 16S rRNA gene. Fractions (1–12) were obtained from density-revealed gradients of the labeling treatment (+13C) or the control (+12C). Increased band intensity was observed in heavy fractions of the labeling treatment. (b) The DGGE profile of bacterial 16S rRNA genes in gradients of the labeling treatment and (c) the control. Arrows indicate noticeable changes in community composition of the labeling treatment.(TIF)Click here for additional data file.Figure S4Fungi and archaea detection in the gut of Spodoptera littoralis. Gel electrophoresis shows the absence of amplification products with fungus- and archaea-specific primers. Three kinds of common fungi-growing agar plates  were used in the fungal cultivation attempt and no fungus was recovered.(TIF)Click here for additional data file.Figure S5Images of Enterococcus sp. from S. littoralis reveal bacterial gut localization and antimicrobial activity. (a) FISH with a Cy3-labeled Enterococcus-specific probe (yellow) show a high density of bacterial cells adhere on the mucus layer lining the gut epithelium, and that under higher magnification (63×10) (b). White arrow indicates the gut epithelium tissue. White arrowhead indicates the gut lumen. (c) Agar diffusion assays show Enterococcus culture filtrates against Micrococcus luteus and (d) Leuconostoc mesenteroides. Antimicrobial activity is detected by the formation of clearance zone around the loading hole.(TIF)Click here for additional data file.Figure S6The gut microflora of S. littoralis larvae fed on the white bean diet. The metagenomic DNA was extracted from the whole gut tissue and sequenced without the cesium gradient ultracentrifugation.(TIF)Click here for additional data file.


Ostrinia furnalalis. Total 14690 stage specific genes were obtained which can be considered as potential targets, and 47 were confirmed by qRT-PCR. Ten larval stage specific expression genes were selected for RNAi test. When 50 ng/µl dsRNAs of the genes DS10 and DS28 were directly sprayed on the newly hatched larvae which placed on the filter paper, the larval mortalities were around 40∼50%, while the dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities reached 73% to 100% at 5 d after treatment. The qRT-PCR analysis verified the correlation between larval mortality and the down-regulation of the target gene expression. Topically applied fluorescent dsRNA confirmed that dsRNA did penetrate the body wall and circulate in the body cavity. It seems likely that the combination of DGE-tag with RNA-seq is a rapid, high-throughput, cost less and an easy way to select the candidate target genes for RNAi. More importantly, it demonstrated that dsRNAs are able to penetrate the integument and cause larval developmental stunt and/or death in a lepidopteron insect. This finding largely broadens the target selection for RNAi from just gut-specific genes to the targets in whole insects and may lead to new strategies for designing RNAi-based technology against insect damage.The key of RNAi approach success for potential insect pest control is mainly dependent on careful target selection and a convenient delivery system. We adopted second-generation sequencing technology to screen RNAi targets. Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies were used to screen optimal RNAi targets from  RNA interference (RNAi) refers to double-stranded RNA (dsRNA) mediated gene silencing Caenorhabditis elegansThe dsRNA is delivered either by injection or by feeding insects. It has been reported that in several insect orders the gene can be knocked down by dsRNA injection de novo reconstruction of the transcriptome for a nonmodel organism Regarding the optimal target gene selection, taking advantage of the known genes and cDNA library screening were the main ways up to now Ostrinia furnalalis (Guenée) (ACB) is a major pest of corn in eastern and south-eastern Asia. At present, its control relies mainly on insecticides. Although the transgenic crops producing Bacillus thuringiensis (Bt) toxins for the pest insect control have been succeeful, their efficacy was gradually reduced when pests evolved resistance Asian corn borer In this study we report that RNAi-induced lethality could be achieved by direct delivery via the exoskeleton; moreover an effective high-throughput NGS method was used to select stage-specific RNAi target genes in the Asian corn borer.de novo assembled into 124,034 contigs with the assemble software of SOAPdenovoTo obtain a global view of transcriptom in the whole ACB life cycle, high-throughput RNA-seq was performed by Solexa/Illumina sequencing technology in Beijing Genomics Institute at Shenzhen. We obtained more than 33 million paired-end reads of 75 bp in length. The total lengths of the reads were over 2.5 giganbases (Gb). The clean reads were We sequenced four DGE-tag libraries from four ACB developmental stages of egg, larva, pupa and adult. After eliminating low quality tags (containing Ns), copy numbers less than 2 and adaptor sequences, the numbers of total clean tags were about 3.57, 3.54, 3.55 and 3.45 millions for eggs, larvae, pupae and adults, respectively. The four successive developmental stages possessed 71004, 71228, 89637 and 89116 clean tag sequences, respectively. These tag sequences were composed of the DGE-tag datasets presented in To obtain the profile of the differential expression genes, the distribution of tag copy numbers in each of the four stages, is summarized in At first, we selected 47 larval stage high expression tags to determine their expression levels by qRT-PCR. All of them indeed appear consistent with their DGE-tag copy numbers . To screin vitro synthesized dsRNAs  ng/µl for DS10 and 60.44 ng/µl  for DS28. This indicates that dsRNA can be delivered into haemolymph through the exoskeleton and still causes insect mortality. Another evidence is produced using labeled fluorescent dsRNA (see below).To explore a convenient way of dsRNA delivery, 10 d dsRNAs  were sprd dsRNAs ; Table 6d dsRNAs , LC50 weTo investigate the correlation of larval mortality with the target gene expression, the expression levels of all 10 genes were measured by qRT-PCR after spraying 50 ng/µl dsRNA. The expression levels of the 10 genes increased 1 day after dsRNA treatment , red barWhen 20 ng/µl dsRNA solution was sprayed on the larvae along with the artificial diet, the larval development was remained in the first instar until 7 days after treatment and a certain degree of larval death was seen when the control larvae had reached the third instar stage . When ACTo further confirm that dsRNA can penetrate the egg shell and the larval body wall of ACB, a fluorescent nucleotide was added during the dsRNA synthesizing of gene DS12. When 0.5 µl of 5 µg/µl fluorescent labeled dsRNA was topically applied on the larval protergum of the matured last instar larvae, fluorescence showed that penetration through the larval cuticle took only a few minutes and in 4 hours the whole body was reached . After 2et al. (2009) summarized the advantages of RNA-seq et al. (2009) comparing DGE-tag with the microarray technology, emphasized that this method has a high sensitivity and reproducibility for transcriptome profiling Taking advantage of known genes is a simple and effective way to verify RNAi targets, but the scope of selection was limited et al. (2011). As indicated in this review, the majority of RNAi studies in Lpidoptera were done using Bombyx mori, Manduca sexta and some Noctuidae species. Systemic RNAi has been demonstrated in some species, such as Hyalophora cecropia and B. mori, in which injection of dsRNA into the pupa can result in phenotypic effects in developing embryos, indicating dsRNA uptake by the developing oocytes of the pupa. However, a great variation of sensitivity to systemic RNAi has been seen among different lepidopteran species and high or no silencing can occur at very different concentrations of dsRNA AaeIAP1 dsRNA products in acetone dilution was able to kill adult female mosquito Aedes aegyptiMaduca sextaHelicopverpa armigeraB. moriSuccessful and unsuccessful RNAi experiments have been reviewed in a number of lepidopteran species by Terenius In conclusion, our results demonstrate that a direct delivery via integument is a feasible way to introduce dsRNA into the haemolymph, which then leads to a down-regulation of gene expressions and finally to the development retarding and/or death. However, some questions still remain to be further investigated, such as in which tissues or cells these genes are silenced and whether the proposed receptor mediated endocytosis or the transmembrane channel-mediated uptake are the mechanisms leading to the production of RNAi effect as well as the pathway of RNAi effect leading to larval developmental stunt and death in ACB larvae. Nevertheless, our findings considerably broaden the selection of targets for RNAi research from gut-specific to whole insects. It may lead to new strategies in designing the RNAi-based technology against insect damage.Ostrinia furnacalis (Guenée) eggs were obtained from Sun Yat-Sen University and reared in laboratory at 25°C and 75% relative humidity on a 14-h-day/10-h-night cycle. Larvae were fed on a modified artificial diet .Asian corn borer 24 samples were collected from egg to adult stages during the whole life cycle of the ACB. The samples were immediately frozen in liquid nitrogen, and stored at −80°C before RNA extraction. Total RNA was isolated using a Qiagen RNA Extraction kit according to the manufacturer's instructions. It was treated with RNase-free DNase I for 30 min at 37°C (New England BioLabs) to remove residual DNA. Then the equivalent amount of the 24 samples was merged into 4 pools of egg, larva, pupa and adult. The total RNA in the whole life cycle came from the 4 pools in equivalent amounts. mRNA was isolated from DNA-free total RNA using Dynabeads mRNA Purification Kit (Invitrogen).18 primer and reverse transcriptase (Invitrogen). The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). Then the 100–500 bp paired-end cDNA library was constructed for sequencing using the Illumina/Solexa technology.First-strand cDNA was synthesized using Oligo(dT)de novo assembled using SOAPdenovo program Short read sequences were Sequence tag was prepared with Illumina's Digital Gene Expression Tag Profiling Kit according to the manufacturer's protocol. For the readers who want to have a schematic overview of the procedure, please see the reference We filtered out low quality tags (containing Ns), copy number below 2 and adaptor sequences. At last, we obtained 3.4∼3.5 million clean sequence DGE-tags for each developmental stages of egg, larva, pupa and adult. The DGE tags, which consist of the CATG restriction enzyme digested site and an additional 17 bp from each transcript, were aligned onto the transcriptome unigenes of ACB. The expression abundance of transcripts was measured by the number of tags mapped.dsRNAs were synthesized using the MEGAscript RNAi kit  according to manufacturer's instructions. T7 promoter sequences were tailed to each 5′ end of DNA template by PCR amplifications. Double stranded EYFP (dsEYFP) was generated using pPigbacA3EYFP as template and was used as a negative control in the experiments. All the primer sequences were listed in The dsRNA synthesis procedure was the same as described above except that 0.25 µl 10 mM fluorescent dCTP labeled with Cy3 was added. Taking advantage of character, T7 RNA polymerase can take dNTP as substrates for synthesis RNA 50 was calculated by using 5 different concentrations, 20, 40, 60, 80, 100 ng/ul dsRNA. The data were analyzed using one-way ANOVA to look for treatment effects compared to the untreated control (P<0.05). In addition, the egg masses were soaked in 50 ng/µl dsRNA soaking buffer for 2 hours and the effect on embryo development and the hatching rate was examined Three hundred newly hatched larvae were placed in a culture dish (9 cm in diameter). 300 µl 50 ng/µl dsRNA in dsRNA buffer tC values obtained for each gene. qRT-PCR was carried out using a Mastercycler ep realplex instrument (Eppendorf). Data analysis methods were described as the reference Expression levels of 10 larval stage specific genes selected according to the DGE-tag copy number were evaluated by qRT-PCR. Primer sequences were provided in To assess the extent of RNAi, RNA was extracted from pools of 20∼30 dsRNA-treated and surviving larvae using a Qiagen RNA Extraction kit. Primer sequences were provided in Table S1DGE-tag mapping by RNA-seq dataset and annotated.(XLS)Click here for additional data file.Table S2Larval stage high expression tags copy number confirmation by qRT-PCR.(XLS)Click here for additional data file.


P<0.05). The site with near-to-nature forest management (low forest management intensity) exhibited no significant differences in litter decomposition rate, C release, lignin decomposition, and C/N, lignin/N and ligninolytic enzyme patterns compared to the unmanaged deciduous forest reserves, but most nutrient dynamics examined in this study were significantly faster under such near-to-nature forest management practices. Analyzing the activities of ligninolytic enzymes provided evidence that different forest system management practices affect litter decomposition by changing microbial enzyme activities, at least over the investigated time frame of 473 days , P = 0.0260). Our results also indicate that lignin decomposition is the rate limiting step in leaf litter decomposition and that MnP is one of the key oxidative enzymes of litter degradation. We demonstrate here that forest system management practices can significantly affect important ecological processes and services such as decomposition and nutrient cycling.Leaf litter decomposition is the key ecological process that determines the sustainability of managed forest ecosystems, however very few studies hitherto have investigated this process with respect to silvicultural management practices. The aims of the present study were to investigate the effects of forest management practices on leaf litter decomposition rates, nutrient dynamics  and the activity of ligninolytic enzymes. We approached these questions using a 473 day long litterbag experiment. We found that age-class beech and spruce forests (high forest management intensity) had significantly higher decomposition rates and nutrient release (most nutrients) than unmanaged deciduous forest reserves ( Almost one-third of the world’s total forest area (ca. 1.2 billion ha) is managed primarily for wood biomass production Although forest system management practices have the potential to affect most characteristics, compositions and structures of the entire forest ecosystem, effects on ecological processes in soils are still unclear Decomposition of plant litter is a complex ecological process that is regulated by three main drivers: climate, litter quality and the decomposer communities In an intensively managed forest, large fractions of woody biomass are normally removed or harvested, thus leaf litter fall remains the main source of organic plant matter input into forest soil In this study, we aimed to examine mixed deciduous leaf litter decomposition rates and the dynamics of macronutrients  under different forest system management practices, including natural, near-to-nature, age-class European beech dominated deciduous forests and converted forests (from European beech to monoculture Norway spruce) by means of a litterbag experiment lasting 473 days. We also investigated the activity of microbial ligninolytic enzymes and examined whether they can help to explain the leaf litter decomposition rates and nutrient dynamics under different forest system management practices. We hypothesized that the decomposition rate of deciduous litter is lower in spruce forest than in other deciduous beech dominated forests Field work permits were issued by the responsible environmental offices of the Free State of Thuringia (according to § 72 BbgNatSchG).2; 51°16’N 10°47’E), part of the German Biodiversity Exploratories Fagus sylvatica dominated) are dominant in this area, covering about 83% of the total forest area. These forests can be classified, according to management types, as unmanaged forest reserves, farmers’ forests, selectively cut forests, and age-class forests Picea abies) r, the risk of stand loss, function of tree species and stand age) and a density component  The study was carried out at four forest sites across the Hainich-Dün region in Central Germany, including the Hainich National Park and its surroundings;  elemental analyzer (DIN/ISO 10694 (Aug. 1996)). Nutrient ions were determined using inductively coupled plasma (ICP) optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS) according to manufacturers’ specifications. The ICP-OES device “Optima 3000”  was calibrated using “ICP multi-element standard solution IV”  as well as “phosphorus ICP standard”  in 1 mg/L and 10 mg/L concentrations. The ICP-MS “Elan DRC-e” (PerkinElmer) device was calibrated using “ICP multi-element standard solution VI”  in 2 μg/L, 15 μg/L and 50 μg/L concentrations. Samples were derived from filtered , aqueous extracts of 10 g coarsely ground (on dry ice) litter material from one replicate. The mean values for the three biological replicates from each sampling point were calculated and presented as μg per gram of litter dry mass. Extractions and ion determinations were undertaken once for each biological replicate, dry mass determination was performed three times.2SO4Total lignin was obtained by summing Klason lignin (acid insoluble lignin) and acid soluble lignin Aliquots of all samples from 89 to 473 DAI were used to determine ligninolytic enzyme activities. Three important enzymes were chosen: laccase (EC 1.10.3.2), peroxidase (EC 1.11.1.7) and manganese peroxidase  420 = 36 mM−1cm−1) in a sequential assay in 50 mM sodium citrate buffer at pH 4.5 . Measurement started with the addition of ABTS, the ABTS oxidation without H2O2 (laccase activity) was recorded for some time, before H2O2 was added; the peroxidase activities were corrected by those of laccase.Laccase and peroxidase activities were spectrophotometrically determined in 1 mL cuvettes by following the oxidation of ABTS  at 420 nm . Measurement started when H2O2 was added.Manganese peroxidase activities were specifically assayed as described previously by monitoring the formation of Mn−1 under the assay conditions used; enzyme activities are given per gram of dry mass of the particular litter sample. For each extract, enzyme activity and dry mass were determined three times. All chemicals used were purchased from Sigma-Aldrich  and Merck .Samples were derived from 10-fold concentrated  aqueous extracts of 10 g coarsely ground (on dry ice) litter material from one replicate. In all cases, the mean values of triplicate determinations were calculated and expressed in international units (U) defined as the amount of enzyme that forms 1 mmol product mink) for each forest type (treatment) were determined by fitting a single negative exponential decay equation Total C and N were expressed as a percentage of the initial value, based on ash-free dried mass. Nutrient ions  were expressed as percentage of initial values based on dry litter mass. The expression of the N, Mg, K, Ca and P contents in the leaf litter as a percentage of the initial content allowed us to determine the net N, Mg, K, Ca and P dynamics X = dried mass remaining at time t, X0 = original mass, “e” = the base of natural logarithms, k = decomposition rate, and t = time (year). All data were analyzed using a one-way analysis of variance (ANOVA) incorporating the Fisher’s Least Significant Difference (LSD) implemented in SPSS software  to determine the differences (P<0.05) between different treatments at each DAI. All datasets were tested for normality using Shapiro-Wilk and Jarque-Bera tests and the equality of group variances was examined using Levene’s test. ANOVA residuals were also plotted to determine the normal probability using Shapiro-Wilk test. In addition, ligninolytic enzyme activities were also analyzed using two-way (forest system management practice and DAI) ANOVA with P<0.05 considered significant, implemented in the PAST program where F = 10.37, P = 0.0039). At the end of the experiment, SA and BA had significantly lower mass remaining (higher decomposition rates) than BU  (The remaining mass of leaf litter varied significantly with forest system management practice (treatments). Surprisingly, we did not find a negative effect of high forest management intensity and/or conversion of forest types on the leaf litter decomposition rate for any of the DAI sampling times. In general, leaf litter in unmanaged beech forest (BU) decomposed relatively slower than in other forests, and the results were significant at 362 and 473 DAI . One-way 0.0378) ; Table 3Leaf litter from BU had lower amounts of N and higher C/N and total lignin/N ratios compared with other forest management practices . InitialF = 1.25, P = 0.3545). The BA forest had a significantly higher C/N ratio in leaf litter compared with other forests from 284 DAI until the end of the experiment  were significantly influenced by forest system management practice. The changes in the amounts of different nutrients relative to their initial values, C/N and total lignin/N ratios over 473 days of incubation are shown in periment .P = 0.0500 and 0.0518) and at 180, 284 and 362 DAI . Large fractions of lignin in the SA and BA forests were decomposed earlier than in the BU and BS forests; for example, at 89 and 180 DAI, SA and BA forest had lost 29–30% of Klason and total lignin while BU and BS forests reached this level around 362 DAI  in leaf litter relative to the initial values were significantly affected by forest system management practices. Patterns of lignin decomposition  between BU and BS (low forest management intensity) forests, and between BA and SA (high management intensity) forests were similar across the DAI sampling times . Interes 362 DAI . The Kla 362 DAI .F = 14.00; P<0.0001) and MnP  activities, while the effects of DAI and the interaction between forest system management practice and DAI were not significant , whilst the effects of forest system management practices and the interaction between forest types and time were not significant  of leaf litter from an additional one or two tree species in BA and BS forest compared with BU forest could dramatically change the decomposition rate (31–72%). There is some evidence that forest management practices not only change tree species richness but also the microclimate and the microbial decomposer community The higher mass loss and net nutrient release (for most of nutrients) in the BA forest as compared with the BU forest may be due to changes in the soil microbial community structure and activity induced by different forest system management practices, as reported in a recent soil fungal diversity study carried out at the same study sites When comparing SA and beech dominated forests, we found no significant home-field advantage. This is because the SA forest in this experiment is accustomed to periodic deciduous litter deposits. In our study, we were able to sample deciduous litter on this site simply because some deciduous trees (∼20%) have established here as a result of natural regeneration. We also postulate that because decomposer communities in SA forest can cope with more recalcitrant substances, such as resins, polyphenols and guaiacyl lignin found in spruce needles, they may also have the capacity to decompose the main deciduous leaf litter (European beech), which is also considered to be a low quality litter, quite rapidly In this study, we demonstrated that the decomposition of complex substances such as lignin was not negatively affected by forest system management practices and/or the naturalness of the forest ecosystems. We found that semi-natural and artificial systems (BA and SA forests) are more efficient at decomposing lignin than near natural (BS forest) and natural systems (BU forest). In addition, the percentage of remaining C in the leaf litter in BU forests was high (indicating slow decomposition) compared with other forest system management practices at all sampling times, and at the end of the experiment there was no significant difference in remaining C in different forest management types. Since no significantly negative effects of forest system management practices were found for either lignin or C decomposition, we can conclude that C cycling was not negatively influenced by intensive forest management, although we could not quantify the exact amounts of leaf litter C release to the forest soil, since litter C can be released both to the soil and to the atmosphere 2+  attacking preferentially phenolic lignin structures which leads to the formation of instable free radicals that tend to disintegrate spontaneously The ligninolytic enzyme activities recorded indicate that forest system management practices control the litter decomposition rates via changes in microbial, in the first place fungal, activities. Although both laccase (high activity in BU and BS forests) and MnP (high activity in BA and SA) can catalyze the oxidation of phenolic lignin moieties, laccase has a generally lower oxidative strength and redox potential compared to peroxidases In our experiment, we found that the differences in management may affect litter decomposition by altering the composition of the vegetation. To improve our mechanistical understanding of this point, a common garden experiment where all litters are incubated in one place may be helpful. However, litter decomposition is not only depending on the litter material used but is also strongly impacted by the microclimatic and other environmental conditions  We conclude that forest system management practices can significantly influence leaf litter decomposition rates and nutrient dynamics in Central European forests. The effects of forest system management practices on litter decomposition rate and nutrient dynamics in beech dominated forests  are complex and are controlled by a range of factors, including tree diversity, microclimatic conditions and the microbial decomposer community. In managed forest ecosystems where a large fraction of woody biomass is harvested, as is the case in BA and BS forests, the system can balance the nutrient status by increasing leaf litter decomposition rates and nutrient dynamics.


Using an olaparib sensitization screen in a triple-negative (TN) breast cancer model, we identified nicotinamide phosphoribosyltransferase (NAMPT) as a non-redundant modifier of olaparib response. NAMPT is a rate-limiting enzyme involved in the generation of the PARP substrate β-NAD+ and the suppression of β-NAD+ levels by NAMPT inhibition most likely explains these observations. Importantly, the combination of a NAMPT small molecule inhibitor, FK866, with olaparib inhibited TN breast tumour growth in vivo to a greater extent than either single agent alone suggesting that assessing NAMPT/PARP inhibitor combinations for the treatment of TN breast cancer may be warranted.PARP inhibitors have been proposed as a potential targeted therapy for patients with triple-negative  breast cancers. However, it is as yet unclear as to whether single agent or combination therapy using PARP inhibitors would be most beneficial. To better understand the mechanisms that determine the response to PARP inhibitors, we investigated whether enzymes involved in metabolism of the PARP substrate, β-NAD Of the 17 or so PARP superfamily members, PARP1 is the most studied, and is known, amongst other functions, to play a critical role in DNA repair. In this role, PARP1 binds damaged DNA and this interaction stimulates auto-PARsylation of PARP1 and trans-PARsylation of additional DNA repair mediators such as XRCC1 and chromatin structure proteins such as histones. Ultimately, these PARSylation events orchestrate a molecular cascade that results in the repair of DNA lesions such as single-strand DNA breaks  chains onto proteins (PARsylation) is a post-translational modification that has been implicated in a wide range of biological processes as diverse as the maintenance of genomic stability  gene defects (such as loss-of-function BRCA1 or BRCA2 mutations), suggests that these molecules could have some utility as single agents .To further assess the generality of our observations, we addressed the possibility that NAMPT inhibition could also increase the therapeutic effect of olaparib in additional TN breast cancer cell lines. FK866 exposure caused clear olaparib sensitivity in four out of five TN models  as the major source of β-NAD+ for PARsylation reactions is via nicotinamide salvage and the activity of NAMPT, non-competitive inhibition of NAMPT  could limit β-NAD+ levels, reduce the extent of β-NAD+/PARP inhibitor competition for the PARP catalytic domain and thus exacerbate the deleterious effects of PARP inhibitors on cells.In light of these observations, we propose the following scenario to explain the synthetic lethality between PARP and NAMPT inhibition: (i) drugs such as olaparib cause cell inhibition by causing persistent DNA lesions and/or impairing DNA repair; (ii) as olaparib is a reversible catalytic inhibitor that competes with β-NADi.e. the detection of nuclear γH2AX foci), is therefore routinely used to estimate the extent of DNA damage caused by PARP inhibition  a cohort treated with daily olaparib, (ii) a cohort treated with FK866, (iii) a cohort treated with an olaparib/FK866 combination and (iv) a vehicle-treated cohort. Neither single agent olaparib nor FK866 alone suppressed the growth of TN xenografts when compared to the vehicle treatment  showed complete tumour regression by day 39 of treatment with no measurable tumour present at the end of the study, with tumours in the other animals in this cohort exhibiting ostensible disease stabilization. Although xenograft studies such as these are able to generate proof-of-concept data that a human tumour cell can be inhibited in vivo, they are relatively limited in their ability to model many forms of clinical toxicity, including those seen in some patients treated with PARP inhibitors such as olaparib. As such it is not clear whether long-term treatment with an NAMPT inhibitor and a PARP inhibitor would lead to deleterious side effects in a clinical setting. Nevertheless, we did note that in the xenograft experiment, each of the treatment regimes was equally well tolerated, with none of the mice showing a significant change in body weight  and NAMPT inhibitor FK866  were used as previously described  of siRNA for gene X = (median centred data from olaparib-treated wells for gene X) − (median centred data from DMSO-treated wells for gene X). Calculation of the median absolute deviation (MAD) was used to estimate the variance of the DE data. Standardized Z scores for DE were calculated using DE values, the median DE from the entire library and the MAD as variables. A similar analysis was conducted to estimate the effect of each siRNA upon cell viability in the absence of Olaparib and siRNAs that caused significant cell inhibition alone (Z < −3) were excluded from the final analysis. In total, we used data from three biological replicate screens in the final analysis.All cell lines were obtained from American Type Culture Collection (ATCC) and Horizon Discovery Ltd. and maintained according to the supplier's instructions. Cell lines were transfected with SMARTpool siRNAs, using Lipofectamine 2000 (Invitrogen) transfection reagent. The siRNA library (siARRAY — targeting 44 known and putative human PARPS and proteins with an established role in NAD metabolism) was purchased from Dharmacon. Each well in this library contained a SMARTpool of four distinct siRNA species targeting different sequences of the target transcript. Each plate was supplemented with siCONTROL . RNAi screening conditions were optimized and used as described elsewhere . SFs were calculated and drug sensitivity curves plotted as previously described  was used to assess caspase 3/7 activity as per the manufacturer's intructions. CAL51 cells were plated in 96-well plates at the density of 5000 cells per well. Twenty-four hours post-seeding, drug treatment was initiated and maintained for 48 h prior to caspase 3/7 analysis. Annexin V immunostaining and FACS analysis was performed as previously described  and immunoblotted overnight at 4°C with antibodies targeting the following: NAMPT , ACTIN (Santa Cruz Biotech), pH2AX (Millipore) and H2AX (Abnova). Incubation with primary antibody was followed by incubation with a horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection of proteins .Whole-cell protein extracts were prepared from cells lysed in NP250 buffer ; supplemented with protease inhibitor cocktail tablets . Protein concentrations were measured using BioRad Protein Assay Reagent . For Western blot analysis, 50 µg of whole cell lysates were electrophoresed on Novex 4–12% gradient Nuclear γH2AX foci were visualized and quantified by confocal microscopy as previously described  according to manufacturer's instructions.GAPDH endogenous control, and adjusted relative to expression in siCONT cDNA. Samples were quantified in triplicate.Total RNA from CAL51 cell lines was extracted using Trizol (Invitrogen) according to manufacturer's instructions. cDNA was synthesized using Omniscript Reverse Transcriptase System for RT-PCR (Qiagen) with oligo dT as per manufacturer's instructions. Assay-on-demand primer/probe sets were purchased from Applied Biosystems. Real-Time qPCR was performed on the 790DHT Fast Real-Time PCR System (Applied Biosystems). Gene expression was calculated relative to expression of 6 cells per graft). Once tumours were palpable (>20 mm3), mice with similar volume tumours were collated and randomized into four cohorts, . After randomization, drug dosing was initiated. For olaparib, we administered a 15 mg/kg dose to each animal, once daily, a treatment regime known to elicit BRCA2 selectivity  Act 1986 and in line with a UK Home Office approved project licence held by AA.CAL51 cells were mixed 2:1 in matrigel (BD Biosciences) and then injected into bilateral flanks of 6–8 week old female athymic nude mice  in the Graphpad Prism software package.Data from the siRNA screen was processed as described as in the main text and also in (Lord et al,


However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility.The use of cytoplasmic male sterility (CMS) in Fde novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.We generated approximately 53 million sequencing reads and assembled Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations. Capsicum annuum L.) is a member of the Solanaceae family. Originated from South America, it has become an economically significant vegetable and an agriculturally important plant worldwide, particularly in China and Korea Chili pepper (restorer-of-fertility (Rf) genes Rf genes atp6-2 and orf456Rf genes, previous study has shown that they mainly scatter in chili pepper, but seldom in sweet pepper partial restoration (pr) locus has been developed Cytoplasmic male sterility (CMS), resulted from disturbed mitochondrial–nuclear interaction, was a failure to produce functional pollen that can be suppressed or counteracted by nuclear genes known as Near-isogenic lines (NILs) are a pair of lines with identical genetic backgrounds, except for a region near the target gene The throughput of sequencing has been improved greatly and cycle time of sequencing has been significantly shortened due to the emergence and development of next generation sequencing (NGS) technology. The NGS technology, namely RNA-Seq, is efficient and inexpensive to analyze transcriptome in a comprehensive and in-depth way Chili pepper is a diplont plant (2n = 24) with a reported genome size about 3,753–4,763 million base pairs To characterize the anther transcriptomes of chili pepper and seek genes involved in fertility determination, transcripts from the CMS line 121A and its restorer line 121C were isolated, quantified and sequenced. These transcriptomic sequences were then assembled by Trinity and annotated by BLASTing against public databases. Subsequently, the annotated sequences were clustered into putative functional categories using the Gene Ontology (GO) framework and grouped into pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, differentially abundant unigenes were analyzed, and part of the results was validated by relative RT-PCR and real-time RT-PCR. This study presents the first broadly survey of CMS line and the restorer line in chili pepper with RNA-Seq analysis.ArabidopsisThough criteria for gametophyte development was available in model plant Gene prediction was conducted using the ‘GetORF’ software for the 85,144 unigenes, 84,793 of which contain protein coding sequences. The average length of the remaining 351 unigenes is 233 bp. This may be due to the fact that they are too short that cover only Untranslated Regions (UTR).In total, 27,191 (32%) unigenes were significantly matched to known genes in the public databases . In factConsistent with other reports Capsicum annuum in NCBI and tomato coding sequences (CDS) from ITAG2.3 annotation release in SGN. 3,385 (3.98%) and 4,993 (5.86%) of the unigenes match with the Capsicum annuum ESTs and tomato coding sequences, respectively, and when do the comparison conversely we got a similar result with 3,383 (3.97%) for the Capsicum annuum ESTs and 5,006 (5.88%) for tomato coding sequences.In addition, we compared the pepper transcriptome with all the ESTs of Out of 85,144 assembled unigenes, 35,393 unigenes were classified into 25 KOG categories , among wA total of 9,896 unigenes were assigned to 58 functional groups using GO assignment . In eachFunctional classification and pathway assignment were performed by KEGG . In totaAfter calculation, expression of each unigene was obtained. Using the restorer line as a reference, 4,326 up-regulated unigenes (with higher expressions in the sterile line) and 7,061 down-regulated unigenes (with higher expressions in the restorer line) were identified . ResultsTo evaluate the validity of Illumina analysis and to further assess the patterns of differential gene expression, several unigenes from our sequencing results were selected and detected by RT-PCR and qRT-PCR  with uniAll unigenes showing significant differences in transcript abundance between the two materials were mapped to the GO and KEGG pathway database. Differently expressed unigenes obviously enriched in four GO terms, i.e. “extracellular”, “ion transporter activity”, “lyase activity” and “transporter activity” . Twenty Some genes among the differentially expressed unigenes are associated with molecular pathology, though we collected samples from asymptomatic plants. In addition, it has reported that restorer genotype for male sterile cytoplasm of genetic resources was moderately resistant to phytophthora capsici, which implied some differentially expressed unigenes may be associated with both molecular pathology and restoration of fertility atp6 gene of Polilma CMS atp4 gene atp8 gene of Ogura CMS atp9 gene atp6-2Previous studies proved the sharply decreased content of ATP in the male sterile line orf456 found in pepper coxII gene is closely related to CMS Cytochrome oxidase is the marker enzyme of mitochondrial inner membrane with strong activity. It plays important role in the mitochondrial respiratory chain electron transfer system and affects plant cell respiration. For the 18 cytochrome oxidase-related unigenes found in the present study, 2 and 7 unigenes were highly abundant in 121A and 121C, respectively. Many studies have shown that cytochrome oxidase is relevant to CMS in plants Rfo gene Rf-1 gene Rf-PPR592 gene The PPR (pentatricopeptide repeat) gene family is a large gene family characterized by the presence of tandem arrays of a degenerate 35-amino-acid repeat Since the abnormal development of anther or pollen is the immediate cause of male sterility, proteins related to anther and pollen may have close relationships with fertility. We found 2 anther specific proteins were more abundant in 121C than in 121A. Proteins related to pollen, including major pollen allergen, pollen coat-like protein and pollen-specific protein, were notably represented among 121C transcripts, with 13 highly abundant unigenes compared with 1 abundant unigene in 121A.The abnormity of activated oxygen metabolism in the development of anther or young panicle may be associated with male sterility It's lack of soluble sugar in the male sterile lines, like pepper Insufficiency or deviation of RNA editing may form improper editing products and therefore hinder the proper functions that generate CMS The MADS-box gene family is a regulatory gene family with specific sequence in plants. Proteins coded by this gene family play important roles in regulation of growth and development of plants. It regulates the development of roots, leaves, fruits and flowers The F-box proteins with F-box domain can identify substrates in the ubiquitin-mediated proteolysis pathways, and play important roles in cell cycle, signal transduction, transcription, programmed cell death and male sterility In the present study, we not only profiled the transcriptome of pepper anther, but also analyzed differentially abundant unigenes between a pepper CMS line 121A and its near-isogenic restorer line 121C. The total 5.3 Gb data were assembled into 85,144 unigenes. We assembled 71,576 and 75,920 unigenes from 121A and 121C, respectively. 4,326 and 7,061 unigenes were found highly abundant in lines 121A and 121C, respectively. After further enrichment analysis, we identified three enriched pathways that cover the most differentially abundant unigenes. Our results provide a global look at the differences between the pepper CMS line and its near-isogenic restorer line, and laid the foundation for identifying new fertility-associated genes and elucidating the mechanisms of cytoplasmic male sterility and fertility recovery.The pepper CMS line 121A and its near-isogenic restorer line 121C were selected as materials for RNA sequencing. They were drawn from a backcross program Flower buds were collected from each of the 20 individuals of 121A and that of 121C, respectively. And collected flower buds were divided into five phases according to the relevance between development phases of microspore and morphological characteristics of floral organs  following the standard protocol. The concentration of total RNA was determined by NanoDrop , and the RNA integrity value (RIN) was checked using RNA 6000 Pico LabChip of Agilent 2100 Bioanalyzer . Then RNA of the five phases was equally mixed.The mixed RNA was incubated with DNase I , and messenger RNA was further purified with MicroPoly(A) Purist Kit  as per the protocol and the final concentration was determined using NonoDrop.RNA was fragmented and annealed with Biotinylated Random Primers which have the Illumina adapter sequence. Then the RNA fragments were captured by Strapavidin through Biotinylated Random Primers. Another Illumina adapter was ligased to 5′RNA by RNA ligase. Reverse transcriptase was used for reverse transcription. Finally two double strand Illumina libraries were obtained by PCR amplification.The two libraries were sequenced by Illumina paired-end sequencing technology. Raw data was scanned using Casava with default parameters, and reads with more 10% Q<20 bases were removed. All sequences smaller than 70 bases were eliminated based on the assumption that small reads might represent sequencing artifacts de novo assembled with Trinity, open reading frames were identified by using an in-house developed program based on ‘GetORF’ from EMBOSS −5, and then the best one was chosen as the result of gene annotation. Comparisons with the ESTs of Capsicum annuum in NCBI and tomato CDS in SGN were performed through Perl scripts, with E value less than 10−10 and 10−20, respectively, and the proportion of the similar part larger than 80%. Gene ontology analysis was performed using GoPipe −5, and then the GO information was obtained according to gene2go. The metabolic pathways were constructed based on KEGG database by BBH  method After 2Ratio ≥1” was used as the threshold to judge the significance of unigene expression difference. The data analyzed have been deposited on the NCBI Gene Expression Omnibus under accession no. GSE45431.Reads number of each unigene was firstly transformed into RPKM (Reads per Kilo bases per Million reads) Candidate male fertility-related genes were selected according to their annotation , and theAll unigenes showing significant transcript abundance differences between the two materials were firstly mapped to the GO and KEGG pathway databases, and then the numbers of unigenes for every GO term and KO term were calculated, respectively. Significantly enriched GO and KO terms from the set of differentially abundant unigenes were found using the hypergeometric test, for the sake of comparing these unigenes to the achieved chili pepper transcriptome background. The formula for the gene enrichment test wasactin (GenBank: GQ339766.1) gene was used as the internal control. Expression levels of the unigenes were calculated from the threshold cycle using the delta–delta Ct method Total RNA isolated above was treated with DNase I to remove genomic DNA contamination. The first-strand cDNA synthesis and the qRT-PCR were carried out using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara) and SYBR Premix Ex Taq™(Takara), respectively. The qRT-PCR was performed on an ABI PRISM 7500 Real-Time PCR System with the following cycling parameters: 95°C for 30 s, followed by 40 cycles of: 95°C for 5 s, 60°C 34 s. The Table S1Top BLAST hits from BLASTING Capsicum annuum L. unigenes against public databases.(XLS)Click here for additional data file.Table S2KEGG biochemical mappings for Capsicum annuum L.(XLS)Click here for additional data file.Table S3Differentially expressed unigenes between 121A and 121C.(XLS)Click here for additional data file.Table S4The primer lists for unigenes used for RT-PCR.(XLS)Click here for additional data file.


The surface area of early visual cortices varies several fold across healthy adult humans and is genetically heritable. But the functional consequences of this anatomical variability are still largely unexplored. Here we show that interindividual variability in human visual cortical surface area reflects a tradeoff between sensitivity to visual details and susceptibility to visual context. Specifically, individuals with larger primary visual cortices can discriminate finer orientation differences, whereas individuals with smaller primary visual cortices experience stronger perceptual modulation by global orientation contexts. This anatomically correlated tradeoff between discrimination sensitivity and contextual modulation of orientation perception, however, does not generalize to contrast perception or luminance perception. Neural field simulations based on a scaling of intracortical circuits reproduce our empirical observations. Together our findings reveal a feature-specific shift in the scope of visual perception from context-oriented to detail-oriented with increased visual cortical surface area. Surface area features of developing visual cortices are implicated in visual perception. Song et al. measure visual discrimination sensitivity in humans and find that an increase in visual cortical surface area activity is associated with improved discrimination sensitivity and degraded contextual illusions. The arealization of cerebral cortex is controlled by an interplay between genetic and developmental factors4Here we investigated two different hypotheses. The retinotopically defined surface area of early visual cortices represents variability across individuals in the amount of cortical surface devoted to the same visual field space67910A second hypothesis is that the visual cortical surface area influences perception not just through the scaling of effective stimulus size, but also through the scaling of intracortical connections. When visual cortical surface area increases, physical constraints may prevent intracortical connections from lengthening at the same rate as the expansion of cortical surface, and different neurons within a neural population activated by a visual stimulation will thus be less interconnected121414In primate early visual cortices, a prominent visual feature that has an orderly cortical representation is orientation, whereas contrast and luminance do not have orderly cortical representations6789101617N=45), in orientation, contrast and luminance domains.Our hypotheses predict a tradeoff between visual discrimination sensitivity and contextual modulation magnitude that is associated with variation in visual cortical surface area. Therefore, we first investigated whether such a perceptual tradeoff existed across a large group of healthy adult participants =7, P<10−7) or luminance perception (t(42)=11, P<10−13). Thus, the interindividual tradeoff between discrimination sensitivity and contextual modulation magnitude was not a generic perceptual phenomenon, but was instead observed only in the orientation domain among the three feature domains  we studied.We observed substantial interindividual variability both in discrimination sensitivityN=20). In this second group, the part of early visual cortices that responded to visual field stimulation up to 7.2° eccentricity was delineated using standard phase-encoded retinotopic mappingNext we investigated how the retinotopically defined surface area of human early visual cortices related to visual discrimination sensitivity and contextual modulation magnitude, in orientation, contrast and luminance domains. We used standard phase-encoded retinotopic mappingt(17)=2.3, P<0.05) or luminance perception (t(17)=3.1, P<0.01) did. Similarly, we found that the correlations between orientation perception and visual cortical surface area were specific to V1 and did not generalize to V2 or V3 that had significantly lower correlations (t(17)=2.8, P<0.01; V1 versus V3, t(17)=2.4, P<0.05). Together, these results revealed a selective correlation between the surface area of human V1 and the scope of orientation perception  that did not generalize to the surface area of other early retinotopic cortices or to the perception of luminance and contrast.We found that across participants the retinotopically mapped surface area of primary visual cortex (V1) correlated negatively with orientation discrimination threshold , suggestThese results were replicated in the second group of participants , suggest32Our overall findings, that interindividual increase in V1 surface area was selectively associated with a shift in orientation perception from context-oriented to detail-oriented, support our second hypothesis. It suggests that visual cortical surface area influences visual perception through the scaling of intracortical connections, and an orderly cortical representation of a feature space may be essential for the surface area of this visual cortex to influence of the perception of this visual feature. Indeed, in primate V1, orientation is orderly represented with an unbroken coverage over the cortical surface14Finally, as a proof of concept we used computational simulations to test whether the scaling of intracortical connections in a model based on the functional organization of primate early visual cortices could reproduce our empirical observations. A great number of models exist that capture the characteristics of visual discrimination and contextual modulation, including ‘structural’ models based on cortical circuits373738We modelled the processing of four basic visual features—orientation, contrast, luminance and visual field location. In accordance with the empirical literature, model neurons code contrast in monotonic response function44We simulated the model using stimuli similar to the ones used in our psychophysical experiments. To quantify the model’s visual discrimination sensitivity, we simulated it with a set of stimuli that differed in only a single feature . The activation pattern of the model visual cortex was compared for different stimuli along each feature dimension, where the degree of non-overlap in activation pattern increased with the feature difference between stimuli . Such noThe model simulations revealed a tradeoff between discrimination sensitivity and contextual modulation magnitude that correlated with the surface area of the model visual cortex . This trRecent years have witnessed a growing interest in the correlations between macroscopic cortical anatomy and human behaviour1415The well-defined and measurable structure of human early visual cortices provides an opportunity for exploring the mechanisms through which visual cortical surface area influences perception. Here we tested two possible mechanisms that interindividual variability in visual cortical surface area influences perception through the scaling of effective stimulus size alone or through its interaction with the scaling of intracortical circuits. Although these two hypotheses both predicted a change in the scope of visual perception from global, context-oriented to local, detail-oriented with the increases in visual cortical surface area across individuals, this change is predicted to be generic for the first hypothesis but feature-specific for the second hypothesis. Previous studies on related topics show that the retinotopically defined V1 surface area correlates positively with the sensitivity of visual field location discriminationTo explicitly test our two hypotheses, we studied whether variation in visual cortical surface area was associated with a tradeoff between sensitivity to visual details and susceptibility to visual context for perception of the same visual feature, and whether any such tradeoff was generic or feature-specific. We found that interindividual variability in the V1 surface area was associated with a feature-specific tradeoff between discrimination sensitivity and contextual modulation of orientation perception. Our findings therefore supported our second hypothesis, suggesting that an orderly cortical representation of a visual feature where the distance in cortical space reflects the distance in feature space is essential for the scaling of cortical surface area to influence the perception of this visual feature. Indeed, not all visual features have such orderly cortical representation. Rather, only a limited number can be represented in this orderly fashionFor a deeper understanding, it will be of interest for further studies to test how visual cortical surface area affects perception of other visual features. For example, in addition to orientation, another prominent visual feature that has an orderly cortical representation in human primary visual cortex is ocular dominance50Of equal importance to the correlations between visual cortical surface area and perception is consideration of the interindividual perceptual variability itself. Specifically, as luminance or contrast discrimination and contextual illusion did not exhibit any correlation with visual cortical surface area, the anatomical sources that account for interindividual variability in luminance or contrast perception are of particular interest. Just as neurons in V1 are connected in an orderly fashion according to their orientation preference5254As demonstrated here and elsewhere20One group of 45 healthy volunteers  and a second group of 20 healthy volunteers , with normal or corrected-to-normal vision and no significant neurological history, took part in this study. All participants gave written informed consent. The study was approved by the UCL Ethics Committee.−2; maximum luminance=80.9 cd m−2). Neuroimaging experiments were conducted in a Siemens Trio 3T MRI (magnetic resonance imaging) scanner with a 32-channel head-coil. Structural MRI data were collected using T1-weighted modified driven equilibrium fourier transform (MDEFT) sequence (repetition time (TR)=7.92 ms; echo time (TE)=2.48 ms; flip angle=16°; field of view=256 × 240; 176 slices; resolution=1 × 1 × 1 mm). Functional MRI (fMRI) data were collected using a two-dimensional echo planar imaging (EPI) sequence  for the first group of participants, and a three-dimensional multi-shot EPI sequence  for the second group of participants. Visual stimuli were projected onto a screen (size=28.6 × 21.5 cm) in the back of the scanner and viewed through a mirror on the head coil (viewing distance=72 cm for the first and 85 cm for the second group of participants).Psychophysics experiments were conducted in a darkened room where the only significant source of light was provided by the calibrated cathode ray tube (CRT) computer monitor . The surface area of each visual cortical region  was calculated in FreeSurfer by summing up the surface area of all vertices in that cortical region.The fMRI data were preprocessed in Statistical Parametric Mapping software (SPM8) using slice time correction, realignment, unwarping and coregistration to MRI structural image. Polar-angle maps and eccentricity maps were generated by applying fast fourier transform to fMRI time series of each voxel to extract its phase at the stimulation frequency. F-statistic maps indicating the significance of visual response were calculated by dividing the power at the stimulation frequency by the average power across all frequencies. Polar-angle maps were used to delineate the boundaries between different visual areas . The maps were displayed on inflated cortical surfaces reconstructed using FreeSurfer, and the boundaries between different visual areas were delineated manually according to the mirror reversals in the maps. F-statistic maps were used to delineate the inner and outer edges of the retinotopically mapped part of V1, V2 and V3. The maps were thresholded at a significance level of Visual discrimination threshold and contextual illusion magnitude of orientation, contrast and luminance perception were measured separately in six subexperiments. To disentangle the effects associated with different visual features, we used visual stimuli that each contained information associated with only a single feature. Specifically, uniform gray stimuli that contained no contrast and no orientation information were used in the luminance experiment; low-pass filtered white noise stimuli that contained no orientation information and had constant average luminance were used in the contrast experiment; sinusoidal grating stimuli  that had constant contrast (full contrast) and constant average luminance were used in the orientation experiment. The stimuli were presented on a uniform gray background whose luminance was 50% of the monitor maximum luminance for orientation and contrast experiments, and 20% of the monitor maximum luminance for luminance experiment. To minimize confounding factors that may influence the anatomy–perception relationship differently for different visual features, we applied the same psychophysical paradigms in orientation, contrast and luminance experiments. Specifically, we applied standard 2-up-1-down staircase procedure to measure visual discrimination threshold and standard method of constant stimuli to measure contextual illusion magnitudeTo measure visual discrimination threshold, two circular stimuli  were presented in succession where one stimulus had a constant feature value  and the other a variable feature value. The interval (first or second), where the stimulus with constant feature value appeared, was randomized. Participants made an unspeeded forced choice regarding whether the second stimulus, compared with the first one, was rotated clockwise or anticlockwise (orientation discrimination), had higher or lower contrast (contrast discrimination), or had higher or lower luminance (luminance discrimination). The duration of each stimulus was 300 ms and the interstimulus interval was 500 ms. The orientation, contrast or luminance difference between the two successively presented stimuli was varied in standard 2-up-1-down staircase fashion that assessed the threshold value at which the discrimination performance converged to 70.7% correctTo measure contextual illusion magnitude, two central circular stimuli , one with and one without surrounding annular context , were presented in succession. The duration of each stimulus was 300 ms and the interstimulus interval was 500 ms. The interval (first or second), where the surrounding context appeared, was randomized but counterbalanced. Participants made an unspeeded forced choice regarding whether the central stimulus in the second interval, compared with the one in the first interval, was rotated clockwise or anticlockwise (orientation illusion), had higher or lower contrast (contrast illusion), or had higher or lower luminance (luminance illusion). Before the experiment, each participant performed four trials in which they manually adjusted the orientation, contrast or luminance of the central stimulus presented in isolation till it matched the perceived orientation, contrast or luminance of the central stimulus presented in the surrounding context. In the subsequent experiment, the orientation, contrast or luminance of the central stimulus presented in surrounding context was kept constant, while that of the central stimulus presented in isolation was varied around this previously measured point of perceptual equality for seven different values. In each experiment, a total of 112 trails  were taken to produce a psychometric curve fitted with logistic function. The contextual illusion magnitude was quantified as the feature difference between the two central stimuli at the 50% threshold point of the psychometric curve where they appeared perceptually equal. In the orientation experiment (tilt illusion), the surrounding context was tilted 15° from the central stimulus. In the contrast experiment (contrast–contrast illusion), the surrounding context had a contrast of 100% and the central stimulus a contrast of 40%. In the luminance experiment (simultaneous brightness contrast illusion), the surrounding context was always present, and it was black in one interval, whereas white in the other.On the basis of the functional organization of primate early visual cortices, we built a one-dimensional single-layer neural field model that described how the intracortical circuits modulated the spatiotemporal dynamics of neural activity3841C.S., D.S.S. and G.R. designed the overall study and wrote the paper. C.S. and D.S.S. collected and analysed the fMRI data. C.S. collected and analysed the psychophysics data and designed and built the computational model.How to cite this article: Song, C. et al. Variability in visual cortex size reflects tradeoff between local orientation sensitivity and global orientation modulation. Nat. Commun. 4:2201 doi: 10.1038/ncomms3201 (2013).Supplementary Figure S1, Supplementary Table S1, Supplementary Notes 1-5 and Supplementary References


Likewise, SIRT3 overexpression reduces glycolysis in tumor cells. In sum, a better understanding of sirtuin-mediated regulation may identify novel ways to therapeutically target tumor metabolism.Mitochondrial sirtuins are NAD-dependent enzymes that bind and regulate numerous metabolic pathways within the mitochondria. For example, SIRT3 functions as an NAD-dependent deacetylase that binds and activates numerous oxidative pathways. We have discovered that sirtuins regulate metabolic pathways important in tumor cell metabolism. One hallmark feature of tumor cells is a shift from oxidative to glycolytic metabolism, and this reliance on aerobic glycolysis to support cell growth is known as the Warburg effect. We have discovered that SIRT3 has an additional effect on cellular metabolism by repressing cellular glycolysis through the regulation of HIF1α, a transcription factor that increases gene expression of glycolytic targets. SIRT3 null cells exhibit metabolic and genetic features of the Warburg effect and enhanced tumorigenecity