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The homeobox family is a large and diverse superclass of genes, many of which act as transcription factors that play important roles in tissue differentiation and embryogenesis in animals. The brown planthopper (BPH), Nilaparvata lugens, is the most destructive pest of rice in Asia, and high fecundity contributes significantly to its ecological success in natural and agricultural habits. Here, we identified 94 homeobox genes in BPH, which could be divided into 75 gene families and 9 classes. This number is comparable to the number of homeobox genes found in the honeybee Apis mellifera, but is slightly less than in Drosophila or the red flour beetle Tribolium castaneum. A spatio-temporal analysis indicated that most BPH homeobox genes were expressed in a development and tissue-specific manner, of which 21 genes were highly expressed in ovaries. RNA interference (RNAi)-mediated functional assay showed that 22 homeobox genes were important for nymph development and the nymph to adult transition, whereas 67 genes were dispensable during this process. Fecundity assay showed that knockdown of 13 ovary-biased genes (zfh1, schlank, abd-A, Lim3_2, Lmxb, Prop, ap_1, Not, lab, Hmx, vis, Pknox, and C15) led to the reproductive defect. This is the first comprehensive investigation into homeobox genes in a hemipteran insect and thus helps us to understand the functional significance of homeobox genes in insect reproduction.

S-Adenosyl-l-methionine-dependent methyltransferases (SAMMTases) modulate important cellular and metabolic activities in both prokaryotes and eukaryotes. Here, we functionally characterized an SAMMTase gene (MTase15) in the migratory brown planthopper (BPH), Nilaparvata lugens, which is the most notorious rice pest in Asia. The cDNA sequence of MTase15 is 2764 nt in length with an open reading frame of 1218 nt encoding 405 amino acid residues. Quantitative real-time PCR analysis showed that MTase15 was readily detected from egg to adult stages and extensively distributed in various body parts of adult females and males, with slightly high levels in ovary and testis, respectively. In addition, MTase15 was transcriptionally regulated by the insulin signalling pathway in BPH. RNA-interference-mediated knockdown of MTase15 (dsMtase15) resulted in deficiencies in vitellogenin synthesis and oogenesis, and female infertility. Males with Mtase15 knockdown retained the capability of producing sperms with normal viability, but less sperm was transferred to wild-type (wt) females during copulation, and eggs laid by these wt females arrested embryogenesis. These findings not only assign a functional role to MTase15, but also provide a link between the insulin signalling pathway and epigenetic regulation in BPH reproduction.

A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged–destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates., Author summary The highly conserved insulin/insulin-like growth factor signaling pathway plays a pivotal role in growth, development, and various physiological processes across a wide phylogeny of organisms. Unlike a single InR in the model species such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, most insect lineages have two or even three InR copies. However, the function of the alternative InRs remains elusive. Here, we created a homozygous mutation for a second insulin receptor (InR2) in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens, using the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas9) system. Our findings revealed that InR2 possesses functions distinct from the BPH InR homologue (NlInR1), indicating that multiple InR paralogues may have evolved independently and may have functionally diversified in ways more complex than previously expected in invertebrates.

Simple Summary The brown planthopper (BPH) Nilaparvata lugens can develop into either long-winged or short-winged adults depending on environmental stimuli received during larval stages. The transcription factor NlFoxO serves as a key regulator determining alternative wing morphs in BPH, but the underlying molecular mechanism is largely unknown. Here, we investigated the transcriptomic profile of forewing and hindwing buds across the 5th-instar stage, the wing-morph decision stage. Our results indicated that NlFoxO modulated the developmental plasticity of wing buds mainly by regulating the expression of cell proliferation-associated genes. Abstract The brown planthopper (BPH), Nilaparvata lugens, can develop into either short-winged (SW) or long-winged (LW) adults according to environmental conditions, and has long served as a model organism for exploring the mechanisms of wing polyphenism in insects. The transcription factor NlFoxO acts as a master regulator that directs the development of either SW or LW morphs, but the underlying molecular mechanism is largely unknown. Here, we microinjected SW-destined morphs with double stranded-RNA (dsRNA) targeting NlFoxO (dsNlFoxO) to change them into LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the gene encoding green fluorescence protein (dsGfp) served as a negative control. The forewing and hindwing buds of 5th-instar nymphs collected at 24, 36, and 48 h after eclosion (hAE) were used for RNA sequencing. We obtained a minimum of 43.4 million clean reads from forewing and hindwing buds at a single developmental time. Differentially expressed genes (DEGs) were significantly enriched in various Gene Ontology (GO) terms, including cellular process, binding, and cell part. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that up-regulated genes in dsNlFoxO-treated forewing and hindwing buds were largely associated with the cell cycle and DNA replication. Furthermore, most up-regulated genes displayed higher expression at 24-, and 36-hAE relative to 48 hAE, indicating that wing cells in LW-destined wings might actively proliferate during the first 36 h in 5th-instar nymphs. Our findings indicated that LW development in BPH was likely dependent on the duration of cell proliferation in the 5th-instar stage, which sheds light on the molecular basis of wing polymorphism in insects.

Background A plethora of prognostic biomarkers for esophageal squamous cell carcinoma (ESCC) that have hitherto been reported are challenged with low reproducibility due to high molecular heterogeneity of ESCC. The purpose of this study was to identify the optimal biomarkers for ESCC using machine learning algorithms. Methods Biomarkers related to clinical survival, recurrence or therapeutic response of patients with ESCC were determined through literature database searching. Forty-eight biomarkers linked to recurrence or prognosis of ESCC were used to construct a molecular interaction network based on NetBox and then to identify the functional modules. Publicably available mRNA transcriptome data of ESCC downloaded from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets included GSE53625 and TCGA-ESCC. Five machine learning algorithms, including logical regression (LR), support vector machine (SVM), artificial neural network (ANN), random forest (RF) and XGBoost, were used to develop classifiers for prognostic classification for feature selection. The area under ROC curve (AUC) was used to evaluate the performance of the prognostic classifiers. The importances of identified molecules were ranked by their occurrence frequencies in the prognostic classifiers. Kaplan-Meier survival analysis and log-rank test were performed to determine the statistical significance of overall survival. Results A total of 48 clinically proven molecules associated with ESCC progression were used to construct a molecular interaction network with 3 functional modules comprising 17 component molecules. The 131,071 prognostic classifiers using these 17 molecules were built for each machine learning algorithm. Using the occurrence frequencies in the prognostic classifiers with AUCs greater than the mean value of all 131,071 AUCs to rank importances of these 17 molecules, stratifin encoded by SFN was identified as the optimal prognostic biomarker for ESCC, whose performance was further validated in another 2 independent cohorts. Conclusion The occurrence frequencies across various feature selection approaches reflect the degree of clinical importance and stratifin is an optimal prognostic biomarker for ESCC.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with the ability to bind to a CCAAT box in nearly all eukaryotes. However, the function of NF-Y in the life-history traits of insects is unclear. Here, we identified three NF-Y subunits, NlNF-YA, NlNF-YB, and NlNF-YC, in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens. Spatio-temporal analysis indicated that NlNF-YA, NlNF-YB, and NlNF-YC distributed extensively in various body parts of fourth-instar nymphs, and were highly expressed at the egg stage. RNA interference (RNAi)-mediated silencing showed that knockdown of NlNF-YA, NlNF-YB, or NlNF-YC in third-instar nymphs significantly extended the fifth-instar duration, and decreased nymph-adult molting rate. The addition of 20-hydroxyecdysone could specifically rescue the defect in adult molting caused by NlNF-YARNAi, indicating that NlNF-Y might modulate the ecdysone signaling pathway in the BPH. In addition, NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi led to small and moderately malformed forewings and hindwings, and impaired the normal assembly of indirect flight muscles. Adult BPHs treated with NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi produced fewer eggs, and eggs laid by these BPHs had arrested embryogenesis. These findings deepen our understanding of NF-Y function in hemipteran insects.

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference-based insect pest management.

The brown planthopper Nilaparvata lugens is one of the most destructive insect pests in Asia, demonstrating high fertility and causing huge crop losses by sucking sap of rice as well as transmitting viruses. However, functional genomic studies on N. lugens are seriously constrained by lack of genetic tools. Here, we employed two eye pigmentation genes to generate germ-line mutations in N. lugens using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system. We showed that injection of single guide RNA of the cinnabar gene of N. lugens (Nl-cn ) into pre-blastoderm eggs induced insertion and deletion (indels) in the founder generation (G 0), which were heritably transmitted to the following G1 generation, leading to bright red compound eyes and ocelli. Mutations of N. lugens white (Nl-w ) generated a high mutant rate of up to 27.3%, resulting in mosaic eyes consisting of white and lightly pigmented ommatidia in both G 0 and G1 individuals. The specificity of CRISPR/Cas9-mediated mutagenesis was further bolstered by PCR and RNA interference-based knockdown analysis. These results show that CRISPR/Cas9-mediated gene editing is achievable in a hemipteran insect, offering a valuable tool for the study of functional genomics and pest management in this planthopper species.

To discuss the molecular mechanism of immunoenhancing activities of Hyriopsis cumingii polysaccharides (HCPS), influences of HCPS on mice immunosignaling molecules (IL-2, IL-10, IFN-γ, NO, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)) and T lymphocyte differentiation (to CD4+ and CD8+ T cells) were evaluated by cell model in vitro and/or cyclophosphamide-induced immunosuppression animal model in vivo. Results showed that gastric gavage of crude HCPS could promote the production of IL-2, IFN-γ, inducible nitric oxide synthase (iNOS), cGMP, CD4 and CD8 in a dose-dependent manner. While, crude HCPS by gastric gavage exhibited suppressive effects on the production of IL-10 and cAMP in a dose-dependent manner. Crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) could strengthen peritoneal macrophage expressing iNOS in vitro in a dose-dependent manner. HCPS-stimulated immunostrengthening functions was mediated, at least in part, by immunosignaling molecul...

The target gene of rapamycin (TOR) is conserved from invertebrates to vertebrates, and plays critical roles in cell growth, nutrient sensing, lifespan and reproduction. In this paper, we employed RNA interference (RNAi) to study the function of TOR in male brown planthoppers (BPH), Nilaparvata lugens. Here we discovered that no offspring was produced when wildtype females BPH mated with NlTOR RNAi males. To understand the influence of NlTOR on male BPH infertility, we dissected the reproductive system of a NlTOR RNAi male, and found that the testes were normal and the seminal vesicles were full of sperm, while the accessory gland was poorly developed after knocking down NlTOR expression. Furthermore, transmission electron microscopy data showed defective structure of epithelial cells of the accessory gland, and the deferent duct was collapsed. Also, behavioral data revealed that wildtype virgin females refused to mate with NlTOR RNAi males, and the acoustic signals of dsNlTOR males differed from controls. Our results indicate that NlTOR plays an essential role in the development of the male BPH reproductive system and regulates mating behavior.

Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a ‘master signal’ that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS–FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS–FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements., Author summary Many insects are capable of developing into either long-winged or short-winged adults, but the underlying molecular basis remains largely unknown. Pioneer studies showed that the insulin/insulin-like growth factor signaling pathway acts as a ‘master signal’ that directs wing buds to develop into long or short wings in the wing-dimorphic planthopper, Nilaparvata lugens. However, downstream effectors mediating the IIS pathway effects are unknown. Our findings highlight that vestigial, a key wing-patterning gene, is a main downstream effector that mediates the IIS activity on the development of alternative wing morphs during the wing-morph decision stage. The molecular mechanism of wing formation, including the function of vestigial, has been studied in great depth in the model insect Drosophila melanogaster. Our data provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network in regulating developmental plasticity of wings in insects.

Ion transport peptide (ITP) and its alternatively spliced homologous ITP-like (ITPL) products play important roles in various insect developmental processes. We found for the first time that alternative 5' untranslated regions (5' UTRs) of ITPL (NilluITPLs-1, -2, -3 and -4) control spatiotemporal expression in the brown planthopper, Nilaparvata lugens, as demonstrated by reverse-transcription quantitative PCR. By using an alternative 5' UTR, NilluITPL-1 was expressed exclusively in the male reproductive system, resulting in the production of the NilluITPL seminal fluid protein. Interestingly, NilluITPLs-3 and -4 were expressed exclusively in the integument, indicating a specialized function for NilluITPL during ecdysis and eclosion. We investigated the functions of NilluITP and NilluITPL using RNA interference (RNAi). We did not observe apparent phenotypes when expression of NilluITPLs was suppressed. However, when NilluITP expression was suppressed, the insect exhibited melanism and failed wing expansion, indicating that NilluITP is a neuropeptide associated with wing expansion in addition to bursicon. Additionally, in contrast to bursicon, the insects showed increased melanism when NilluITP was eliminated by RNAi. Unlike previous studies of ITP/ITPL in other species, NilluITP was very important in the control of N. lugens postecdysial behaviours but was not critical during ecdysis. Thus, the functions of ITP and ITPL are more complex in insects than previously thought.

The homeotic complex (Hox) gene Ultrabithorax (Ubx) plays pivotal roles in modifying specific morphological differences among the second (T2), the third thoracic (T3), and the first abdomen (A1) segment in several insects. Whether Ubx regulates wing dimorphism and other morphological traits in the delphacid family (order Hemiptera) remains elusive. In this study, we cloned a full-length Ubx ortholog (NlUbx) from the wing-dimorphic planthopper Nilaparvata lugens, and identified two NlUbx isoforms. RNA-interference (RNAi)-mediated silencing of NlUbx in short-winged BPH nymphs significantly induced the development of wing-like appendages from T3 wingbuds, and this effect is likely mediated by the insulin/insulin-like signaling pathway. RNAi knockdown of NlUbx in long-winged BPH nymphs led to a transformation from hindwings to forewings. Additionally, silencing of NlUbx not only dramatically changed the T3 morphology, but also led to jumping defect of T3 legs. First-instar nymphs derived from parental RNAi had an additional leg-like appendages on A1. These results suggest that Ubx plays a role in determining some morphological traits in delphacid planthoppers, and thus help in understanding evolution of morphological characteristics in arthropods.

Histone acetylation is a specific type of chromatin modification that serves as a key regulatory mechanism for many cellular processes in mammals. However, little is known about its biological function in invertebrates. Here, we identified 12 members of histone deacetylases (NlHDACs) in the brown planthopper (BPH), Nilaparvata lugens . RNAi-mediated silencing assay showed that NlHdac1 , NlHdac3 and NlHdac4 played critical roles in female fertility via regulating ovary maturation or ovipositor development. Silencing of NlHdac1 substantially increased acetylation level of histones H3 and H4 in ovaries, indicating NlHDAC1 is the main histone deacetylase in ovaries of BPH. RNA sequencing (RNA-seq) analysis showed that knockdown of NlHdac1 impaired ovary development via multiple signalling pathways including the TOR pathway. Acoustic recording showed that males with NlHdac1 knockdown failed to make courtship songs, and thus were unacceptable to wild-type females, resulting in unfertilized eggs. Competition mating assay showed that wild-type females overwhelmingly preferred to mate with control males over NlHdac1 -knockdown males. These findings improve our understanding of reproductive strategies controlled by HDACs in insects and provide a potential target for pest control.

Many insects are capable of developing into either long-winged or short-winged (or wingless) morphs, which enables them to rapidly match heterogeneous environments. Thus, the wing polymorphism is an adaptation at the root of their ecological success. Wing polymorphism is orchestrated at various levels, starting with the insect's perception of environmental cues, then signal transduction and signal execution, and ultimately the transmitting of signals into physiological adaption in accordance with the particular morph produced. Juvenile hormone and ecdysteroid pathways have long been proposed to regulate wing polymorphism in insects, but rigorous experimental evidence is lacking. The breakthrough findings of ecdysone receptor regulation on transgenerational wing dimorphism in the aphid Acyrthosiphon pisum and of insulin signaling in the planthopper Nilaparvata lugens greatly broaden our understanding of wing polymorphism at the molecular level. Recently, the advent of high-throughput sequencing coupled with functional genomics provides powerful genetic tools for future insights into the molecular bases underlying wing polymorphism in insects.

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.

The tick Ixodes persulcatus is the predominant tick species in Northeastern China, and it is a major vector in transmission of tick-borne diseases. By 16S rRNA Illumina sequencing, we investigated the microbiome of I. persulcatus and assessed the variation of the microbiome before and after blood feeding. The prolonged blood meal dramatically altered the composition of the microbiome but did not influence the bacterial diversity. Overall, 373 and 289 bacterial genera were assigned to unfed and fed ticks, respectively. To investigate microbes that were potentially transmitted to vertebrate hosts during a blood meal, we examined the microbiome in rat blood after tick bites. Our data showed that 237 bacterial genera were suspected to be pathogens of vertebrates because they were commonly detected in unfed ticks, fed ticks, and rat blood samples after tick bites. Additionally, the prevalence survey on Borrelia burgdorferi s.l., Ehrlichia chaffeensis, Anaplasma phagocytophilum and Yersinia pestis was performed. We found that B. garinii and B. afzelii are the predominant genospecies of the Lyme disease spirochete in I. persulcatus ticks. This is the first time that the microbial composition in this tick species and in rat blood transmitted via tick bites has been reported. These data may ultimately assist in identification of novel pathogens transmitted by I. persulcatus ticks.

Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.

Raltitrexed is a specific inhibitor of thymidylate synthase (TS), which has been considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In the present study, the apoptosis mechanisms of raltitrexed in SGC7901 human gastric cancer cells were investigated. The cytotoxic activity of raltitrexed on SGC7901 cells was determined by cell counting kit-8 (CCK-8) assay. The CCK‑8 assay indicated that raltitrexed inhibits SGC7901 cell growth in a dose- and time-dependent manner. The morphological changes were observed by fluorescent microscopy, and characteristic morphological changes, including nuclear shrinkage and apoptotic bodies, were observed following Hoechst 33258 staining. The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species (ROS) were measured by flow cytometry. The analysis revealed that raltitrexed exerted a growth inhibitory effect by inducing time-dependent apoptosis and cell-cycle arrest at the G0/G1 phase. In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Raltitrexed‑induced caspase‑3‑dependent apoptosis was identified using a caspase-3 activity assay and pretreatment with the caspase-3 inhibitor, Ac‑DEVD‑CHO (sequence, Ac-Asp-Glu-Val-Asp-CHO). The activity of caspase-3 was analyzed with a spectrometer. The protein expression levels of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and TS were examined by western blot and the mRNA expression level of TS was detected by quantitative polymerase chain reaction. The analysis revealed that the protein levels of Bax, cytochrome c and cleaved caspase‑3 were significantly increased by raltitrexed, while Bcl-2 expression levels were reduced. Furthermore, raltitrexed increased the expression of the TS protein and mRNA in a time‑dependent manner. These results indicate that raltitrexed induces the apoptosis of SGC7901 cells through the caspase‑3‑dependent mitochondrial signaling pathway and upregulates the expression of the TS protein and mRNA.

The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings.

The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management.

The brown planthopper (Nilaparvata lugens, BPH), white-backed planthopper (Sogatella furcifera, WBPH) and small brown planthopper (Laodelphax striatellus, SBPH) are important rice pests in Asia. These three species differ in thermal tolerance and exhibit quite different migration and overwintering strategies. To understand the underlying mechanisms, we sequenced and compared the transcriptome of the three species under different temperature treatments. We found that metabolism-, exoskeleton- and chemosensory-related genes were modulated. In high temperature (37 °C), heat shock protein (HSP) genes were the most co-regulated; other genes related with fatty acid metabolism, amino acid metabolism and transportation were also differentially expressed. In low temperature (5 °C), the differences in gene expression of the genes for fatty acid synthesis, transport proteins and cytochrome P450 might explain why SBPH can overwinter in high latitudes, while BPH and WBPH cannot. In addition, other genes related with moulting, and membrane lipid composition might also play roles in resistance to low and high temperatures. Our study illustrates the common responses and different tolerance mechanisms of three rice planthoppers in coping with temperature change, and provides a potential strategy for pest management.

Wing polymorphism contributes significantly to the success of a wide variety of insects. However, its underlying molecular mechanism is less well understood. The migratory planthopper (BPH), Nilaparvata lugens , is one of the most extensively studied insects for wing polymorphism, due to its natural features of short- and long-winged morphs. Using the BPH as an example, we first surveyed the environmental cues that possibly influence wing developmental plasticity. Second, we explained the molecular basis by which two insulin receptors (InR1 and InR2) act as switches to determine alternative wing morphs in the BPH. This finding provides an additional layer of regulatory mechanism underlying wing polymorphism in insects in addition to juvenile hormones. Further, based on a discrete domain structure between InR1 and InR2 across insect species, we discussed the potential roles by which they might contribute to insect polymorphism. Last, we concluded with future directions of disentangling the insulin signalling pathway in the BPH, which serves as an ideal model for studying wing developmental plasticity in insects. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

A baculovirus, named BomaNPV S2, was isolated from a diseased larva of the wild silkworm, Bombyx mandarina. Notably, BomaNPV S2 exhibited a distinguishing feature in that its host range covered that of both Bombyx mori nucleopolyhedrosis virus (BmNPV) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in cultured cells. It could replicate in cells of B. mori (Bm5 and BmN), Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn-5B1-4). However, occlusion-derived virions of BomaNPV S2 in B. mori cells contained only a single nucleocapsid, whereas they contained multiple nucleocapsids in Tn-5B1-4 cells. The complete genome sequence of BomaNPV S2, including predicted ORFs, was determined and compared with the genome sequence of its close relatives. The comparison results showed that most of the BomaNPV S2 genome sequence was shared with BmNPV (BmNPV T3) or BomaNPV S1, but several regions seemed more similar to regions of AcMNPV. This observation might explain why BomaNPV S2 covers the host ranges of BmNPV and AcMNPV. Further recombinant virus infection experiments demonstrated that GP64 plays an important role in BomaNPV S2 host-range determination.

Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family. Most insects possess two CHS genes (CHS1 and CHS2); however, genome and transcriptome searches showed that N. lugens possibly possess only CHS1 in both databases. Two transcript variants (CHS1a and CHS1b) resulting from exclusively alternative splicing (exon 19a or 19b in N. lugens) were identified for each of the two rice planthopper CHS1s. Gene structure comparison using the genomes that are currently sequenced showed that the CHS1 genes in all insects except Acyrthosiphon pisum have two transcript variants. Transcription of NlCHS1a reached its highest level just after molting, whereas NlCHS1b reached its highest expression level 1–2 days before molting. Injection of the N. lugens nymphs with double-strand RNA (dsRNA) of CHS1, CHS1a and CHS1b reduced the corresponding variant transcript levels and exhibited subsequent phenotypes. Silencing of CHS1 and CHS1a resulted in elongated distal wing pads and the “wasp-waisted” or crimpled cuticle phenotypes and eventually died, whereas the phenotypes caused by injection of NlCHS1b dsRNA seem not so obvious although slightly increased mortality was observed. Our results suggest that N. lugens likely lacks CHS2 and CHS1 may be efficient target gene for RNAi-based N. lugens control.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H(2)O(2) and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.

Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.

Dramatic alteration of surface lipoprotein profiles is a key strategy that Borrelia burgdorferi , the Lyme disease pathogen, has evolved for adapting to the diverse environments of arthropod and mammalian hosts. Several of these differentially expressed lipoproteins have been shown to play important roles in the enzootic cycle of B. burgdorferi . The BBA05 protein is a previously identified putative lipoprotein (P55 or S1 antigen) that elicits antibody responses in mammals. Recent microarray analyses indicate that the BBA05 gene is differentially expressed by many environmental factors, including temperature. However, the role of the BBA05 protein in the life cycle of B. burgdorferi has not been elucidated. Here we show that expression of the BBA05 gene was exclusively induced in feeding nymphal ticks during the spirochetal transmission from ticks to mammals. Upon generating a BBA05 mutant in an infectious strain of B. burgdorferi , we showed that the BBA05 mutant remained capable of establishing infection in mice, being acquired by ticks, persisting through tick molting, and reinfecting new mammalian hosts. These results indicate that, despite being a highly conserved and regulated antigen, the BBA05 protein has a nonessential role in the transmission cycle of B. burgdorferi , at least in the animal model.

The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD(50) was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF 51 (Bm51) is a gene present in many lepidopteran NPVs, but its function is unknown. In this study, Bm51 was characterized. Transcripts of Bm51 were detected from 4.5 through 72 hour post infection (h p.i.) by RT-PCR. The corresponding protein was detected from 6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using a polyclonal antibody against Bm51. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm51 encodes a 23-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. The protein was temporarily called BV-E23. In addition immunofluorescence microscopy demonstrated that the protein was present within the cytoplasm and nuclei in virus-infected cells. In conclusion, the available data suggest that Bm51 is a functional ORF of BmNPV and encodes a protein expressed in the early stage of the infection cycle that is associated with the BV envelope.

Alteration of surface lipoprotein profiles is a key strategy that the Lyme disease pathogen, Borrelia burgdorferi , has evolved to be maintained within its enzootic cycle between arthropods and mammals. Accumulated evidence indicates that the central regulatory pathway controlling differential gene expression by B. burgdorferi is the RpoN-RpoS pathway (the σ 54 -σ S sigma factor cascade). It was previously shown that activation of the RpoN-RpoS pathway is controlled by Rrp2, a two-component response regulator and σ 54 -dependent transcriptional activator. The role of Rrp2 in the infectious cycle of B. burgdorferi has not been determined heretofore. In this report, we demonstrate that an rrp2 mutant defective in activating σ 54 -dependent transcription was unable to establish infection in mice, but the rrp2 mutant was capable of surviving within ticks during and after tick feeding. Because the rrp2 mutant was defective in the production of OspC, an outer surface lipoprotein essential for mammalian host infection, we further examined whether the loss of infectivity of the rrp2 mutant was solely due to the inability to produce OspC. While transformation with a shuttle vector carrying ospC under the control of a constitutive flaB promoter restored infection to an ospC mutant in immunodeficient SCID mice, it could not rescue the avirulent phenotype of the rrp2 mutant. These data indicate that, in addition to controlling OspC, Rrp2 controls another factor(s) essential for B. burgdorferi to establish infection in mammals. Furthermore, microarray analyses revealed that 125 and 19 genes were positively and negatively regulated, respectively, by Rrp2, which provides a foundation for future identification of additional Rrp2-dependent virulence determinants in B. burgdorferi .

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of ‘enveloped’ nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.

Most plant viruses that seriously damage agricultural crops are transmitted by insects. However, the mechanisms enabling virus transmission by insect vectors are poorly understood. The brown planthopper (Nilaparvata lugens) is one of the most serious rice pests, causing extensive damage to rice plants by sucking the phloem sap and transmitting viruses, including Rice ragged stunt virus (RRSV). In this study, we investigated the mechanisms of RRSV transmission from its insect vector to the rice plant in vivo using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and RNA interference technology. RRSV induced apoptosis in the salivary gland cells of its insect vector, N. lugens. The RRSV-induced apoptosis was regulated through a caspase-dependent manner and inhibition of the expression of N. lugens caspase-1 genes significantly interfered with virus transmission. Our findings establish a link between virus-associated apoptosis and virus transmission from the insect vector to the host plant.

In the genome of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, open reading frame 39 (Ha39) is the only gene predicted to encode an RNA recognition protein. Computer analysis revealed that Ha39 homologues were found in 15 NPVs, but not in GVs. Its transcripts were detected from 3 through 72 hours post infection (h p.i.) using RT-PCR and Northern blot analysis. The protein was detected in infected-cell lysates from 6 h p.i. Western blot assay of ODV and BV preparations revealed that Ha39 encodes a structural protein associated with BVs.Additionally, immunofluorescence microscopy demonstrated that the protein was present within cytoplasm in virusinfected cells, but not in the nuclear region.

Open reading frame 79 of Bombyx mori nucleopolyhedrovirus (Bm79) is a conserved gene whose homologues have been identified in all 26 of the completely sequenced baculovirus genomes, including lepidopteran NPVs and GVs, hymenopteran NPVs, and a dipteran baculovirus. Northern blot analysis showed that the Bm79 transcript was about 850 nucleotides long and was initiated 12 h p.i. Temporal expression analysis revealed a 28-kDa protein, which was detected beginning 24 h p.i. using a polyclonal antibody against GST-Bm79 fusion protein. The 28-kDa protein was detected in the occlusion-derived virus envelope (ODV-E), but not in budded viruses. This observation was confirmed by observing ultrathin sections of polyhedra using immunoelectron microscopy. This demonstrated that the protein was present within the nuclei of cells. These results suggest that Bm79 is a functional gene that encodes a structural protein associated with the envelope of occlusion-derived virus (ODV).

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) is a single embedded NPV pathogenic to the bollworm, which is a major agricultural pest in many areas around the world. Ha128 homologues have been identified in all completely sequenced lepidopteran NPV's, but no homologue has been found in a granulovirus (GV) and it is thus considered as a lepidopteran NPV-specific gene. In the HearSNPV-C1 genome Ha128 is located between 120,252 and 121,052 bp and encodes a putative protein of 266 amino acid residues with a predicted molecular weight of 30.5 kDa. Ha128 transcripts in HearSNPV-infected HzAM1 cells could be detected from 24 to 120 h post-infection (p.i.) by Northern blot. The Ha128 protein was detected at 24 h p.i. and remained detectable until 120 h p.i. by western blot using an anti-GST-Ha128 antiserum. The expression of Ha128 was inhibited in the presence of Ara-C, an inhibitor of viral DNA synthesis. These results together indicated that Ha128 was a late gene. The product of Ha128 was found to have an Mr of about 31 kDa, in agreement with the predicted molecular weight. Immunoflorescence using anti-GST-Ha128 serum showed that Ha128 was located in cytoplasm. GST pull-down and co-immunoprecipitation showed that at least two potential host proteins interacted with Ha128. In conclusion, Ha128 is a late protein localized in the cytoplasm of infected cells that may interact with host proteins.

Background The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts. Results We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal’s exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host. Conclusions Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0521-0) contains supplementary material, which is available to authorized users.

Flightin was initially identified in Drosophila melanogaster. Previous work has shown that Drosophila flightin plays a key role in indirect flight muscle (IFM) function and has limited expression in the IFM. In this study, we demonstrated that flightin is conserved across the Pancrustacea species, including winged insects, non-winged insects, non-insect hexapods and several crustaceans. The brown planthopper (BPH), Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), a long-distance migration insect with wing dimorphism, is the most destructive rice pest in Asia. We showed that flightin was one of the most differentially expressed genes in macropterous and brachypterous BPH adults. In female BPHs, flightin was expressed in the IFM of macropterous adults, no expression was detected in brachypterous ones; while in male BPHs, flightin was not only expressed in the IFM of macropterous adults, but also in the dorsal longitudinal muscle (DLM) in the basal two abdominal segments of both macropterous and brachypterous ones. RNAi and transmission electron microscopy results showed that flightin played key roles in maintaining IFM and male DLM structure, which drive wing movements in macropterous adults and the vibration of the male-specific tymbal, respectively. Using Daphnia magna as an example of a crustacean species, we observed that flightin was expressed in juvenile instars and adults, and was localized in the antenna muscles. These results illustrate the functional variations of flightin in insects and other arthropod species and provide clues as to how insects with flight apparatuses evolved from ancient pancrustaceans.

DSC1 and BSC1 are two founding members of a novel family of invertebrate voltage-gated cation channels with close structural and evolutionary relationships to voltage-gated sodium and calcium channels. In this study, we searched the published genome sequences for DSC1 orthologs. DSC1 orthologs were found in all 48 insect species, and in other invertebrate species belonging to phyla Mollusca, Cnidaria, Hemichordata and Echinodermata. However, DSC1 orthologs were not found in four arachnid species, Ixodes scapularis, Rhipicephalus microplus, Tetranychus urticae and Varroa destructor, two species in Annelida or any vertebrate species. We then cloned and sequenced NlSC1 and BmSC1 full-length cDNAs from the brown planthopper (Nilaparvata lugens) and the silkworm (Bombyx mori), respectively. NlSC1 and BmSC1 share about 50% identity with DSC1, and the expression of NlSC1 and BmSC1 transcripts was most abundant in the head and antenna in adults. All DSC1 orthologs contain a unique and conserved DEEA motif, instead of the EEEE or EEDD motif in classical calcium channels or the DEKA motif in sodium channels. Phylogenetic analyses revealed that DSC1 and its orthologs form a separate group distinct from the classical voltage-gated sodium and calcium channels and constitute a unique family of cation channels. The DSC1/BSC1-family channels could be potential targets of new and safe insecticides for pest control.

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host., Author Summary The Lyme disease pathogen Borrelia burgdorferi has two sets of two-component systems, Hk1-Rrp1 and Hk2-Rrp2. The Hk2-Rrp2 signaling system has been shown to modulate differential expression of numerous surface lipoprotein genes and to play an essential role in spirochete transformation from a tick colonizer to a mammalian host-adapted state. In this study, we show that Rrp1, the only diguanylate cyclase in B. burgdorferi, is not required for mammalian infection but is essential for spirochete survival in the tick vector. We identify over 39 genes whose expression is influenced by this c-di-GMP signaling system. We further demonstrate that one set of the Rrp1-dependent genes, the glp operon for glycerol transport and metabolism, plays an important role in the spirochete adaptation to tick environment and partially accounts for the essentiality of c-di-GMP for B. burgdorferi survival in ticks.

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The seventh author's name was spelled incorrectly. The correct name is: Frank Gherardini. The affiliation for the third author was incorrect. Tao Lin is not affiliated with #4 but with #5 Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas, United States of America.

Background The brown planthopper (BPH) Nilaparvata lugens (Stal) is one of the most serious insect pests of rice in Asia. However, little is known about the mechanisms responsible for the development, wing dimorphism and sex difference in this species. Genomic information for BPH is currently unavailable, and, therefore, transcriptome and expression profiling data for this species are needed as an important resource to better understand the biological mechanisms of BPH. Methodology/Principal Findings In this study, we performed de novo transcriptome assembly and gene expression analysis using short-read sequencing technology (Illumina) combined with a tag-based digital gene expression (DGE) system. The transcriptome analysis assembles the gene information for different developmental stages, sexes and wing forms of BPH. In addition, we constructed six DGE libraries: eggs, second instar nymphs, fifth instar nymphs, brachypterous female adults, macropterous female adults and macropterous male adults. Illumina sequencing revealed 85,526 unigenes, including 13,102 clusters and 72,424 singletons. Transcriptome sequences larger than 350 bp were subjected to Gene Orthology (GO) and KEGG Orthology (KO) annotations. To analyze the DGE profiling, we mainly compared the gene expression variations between eggs and second instar nymphs; second and fifth instar nymphs; fifth instar nymphs and three types of adults; brachypterous and macropterous female adults as well as macropterous female and male adults. Thousands of genes showed significantly different expression levels based on the various comparisons. And we randomly selected some genes to confirm their altered expression levels by quantitative real-time PCR (qRT-PCR). Conclusions/Significance The obtained BPH transcriptome and DGE profiling data provide comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms from various physiological aspects including development, wing dimorphism and sex difference in BPH.

Borrelia burgdorferi , the Lyme disease pathogen, dramatically alters its protein profile when it is transmitted between ticks and mammals. Several differentially expressed proteins have been shown to be critical for the enzootic cycle of B. burgdorferi . In this study, we demonstrated that expression of the surface lipoprotein-encoding gene bba07 is induced by an elevated temperature and a reduced pH during in vitro cultivation, as well as during nymphal tick feeding. Expression of bba07 is regulated by the Rrp2-RpoN-RpoS pathway, a central regulatory network that is activated during nymphal feeding. By generating a bba07 mutant of an infectious strain of B. burgdorferi , we demonstrated that although BBA07-deficient spirochetes were capable of infecting mice via needle inoculation and surviving in ticks, they were defective in infection of mammals via tick transmission. Complementation of the bba07 mutant with a wild-type copy of bba07 partially restored the transmission defect of the bba07 mutant. Based on these findings, we concluded that the surface lipoprotein BBA07 is produced during tick feeding and facilitates optimal transmission of B. burgdorferi from the tick vector to a mammalian host.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis., Author Summary Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature in a complex enzootic cycle involving Ixodes ticks and mammals. A novel regulatory network, the Rrp2-RpoN-RpoS pathway, which governs differential expression of numerous genes of B. burgdorferi, is essential for this complex life cycle. In this study, we provide evidence showing that the activation of the Rrp2-RpoN-RpoS pathway is modulated, not by the predicted histidine kinase for Rrp2, but rather by acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) metabolic pathway. Based on our findings, we propose that during the enzootic cycle of B. burgdorferi, changes in environmental cues and nutrient conditions lead to an increase in the intracellular acetyl∼P pool in B. burgdorferi, which in turn modulates the activation of the Rrp2-RpoN-RpoS pathway.

I appreciate the mechanism underlying the role of lactate in lipid metabolism proposed by Liu et al. (1). They found that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. However, the argument that lactate suppresses lipolysis might be worthy of further consideration. Several studies have shown that there exists a substrate cycle or futile cycle in skeletal muscle as well as in adipose tissue, which dissipates redundant energy as heat via the cycle of synthesis, hydrolysis, and oxidation of triglyceride (2, 3). Furthermore, Hagstrom-Toft et al. (4) found that hypoglycemia increases both lipid and lactate mobilization in skeletal muscle and adipose tissues. It is well known that hypoglycemia will promote lipid hydrolysis to release fatty acids for providing energy by oxidation. I also found that lactate concentration was positively correlated with glycerol concentration in skeletal muscle and adipose tissues (unpublished data). Therefore, it is possible that lactate produced by anaerobic glycolysis might relieve the inhibition of glucose aerobic oxidation on fatty acid oxidation or adjust intracellular redox state via lactate shuttle in skeletal muscle and adipose tissues and thus promote fatty acid oxidation.

The asymmetric unit of the title compound, C8H6N8·2H2O, contains one half-molecule, with the benzene ring on a centre of symmetry, and two uncoordinated water molecules. The benzene ring is oriented at a dihedral angle of 34.43 (12)° with respect to the tetrazole ring. Strong O—H...N hydrogen bonds link the water molecules to the N atoms of the tetrazole ring. In the crystal structure, strong intermolecular O—H...O and O—H...N hydrogen bonds link the molecules into a network. One of the water H atoms is disordered over two positions and was refined with occupancies of 0.50.

Rrp2 is the sole σ 54 -dependent transcriptional activator present in the Borrelia burgdorferi genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates σ 54 -dependent rpoS expression without an apparent upstream enhancer element commonly associated with other σ 54 -dependent transcriptional activators.