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Description, A feminizing switch in a hemimetabolous insect provides novel insights into the diverse mechanisms of insect sex determination., The mechanism of sex determination remains poorly understood in hemimetabolous insects. Here, in the brown planthopper (BPH), Nilaparvata lugens, a hemipteran rice pest, we identified a feminizing switch or a female determiner (Nlfmd) that encodes a serine/arginine-rich protein. Knockdown of Nlfmd in female nymphs resulted in masculinization of both the somatic morphology and doublesex splicing. The female-specific isoform of Nlfmd, Nlfmd-F, is maternally deposited and zygotically transcribed. Depletion of Nlfmd by maternal RNAi or CRISPR-Cas9 resulted in female-specific embryonic lethality. Knockdown of an hnRNP40 family gene named female determiner 2 (Nlfmd2) also conferred masculinization. In vitro experiments showed that an Nlfmd2 isoform, NlFMD2340, bound the RAAGAA repeat motif in the Nldsx pre-mRNA and formed a protein complex with NlFMD-F to modulate Nldsx splicing, suggesting that NlFMD2 may function as an RNA binding partner of the feminizing switch NlFMD. Our results provide novel insights into the diverse mechanisms of insect sex determination.

The majority of plant viruses are transmitted by hemipteran insects. Bacterial symbionts in hemipteran hosts have a significant impact on the host life, physiology and ecology. Recently, the involvement of bacterial symbionts in hemipteran vector-virus and vector-plant interactions has been documented. Thus, the exploitation and manipulation of bacterial symbionts have great potential for plant viral disease control. Herein, we review the studies performed on the impact of symbiotic bacteria on plant virus transmission, including insect-bacterial symbiont associations, the role of these bacterial symbionts in viral acquisition, stability and release during viral circulation in insect bodies, and in viral vertical transmission. Besides, we prospect further studies aimed to understand tripartite interactions of the virus-symbiotic microorganisms-insect vector.

Simple Summary The bean bug, Riptortus pedestris (Fabricius) is a notorious pest of soybean crops in Asia. During the feeding process, the bug secretes a mixture of salivary components, which play critical roles in the insect–plant interactions. In the present study, a total of 136 salivary proteins were identified by transcriptomic and proteomic approaches. Among them, five proteins (RpSP10.3, RpSP13.4, RpSP13.8, RpSP17.8, and RpSP10.2) were capable of inducing cell death, reactive oxygen species (ROS) burst, and hormone signal changes, indicating the potential roles of these proteins in eliciting plant defenses. Our results provide a good resource for future functional studies of bug salivary effectors and might be useful in pest management. Abstract The bean bug, Riptortus pedestris (Fabricius), is one of the most important soybean pests. It damages soybean leaves and pods with its piercing-sucking mouthparts, causing staygreen-like syndromes in the infested crops. During the feeding process, R. pedestris secretes a mixture of salivary proteins, which play critical roles in the insect–plant interactions and may be responsible for staygreen-like syndromes. The present study aimed to identify the major salivary proteins in R. pedestris saliva by transcriptomic and proteomic approaches, and to screen the proteins that potentially induced plant defense responses. Altogether, 136 salivary proteins were identified, and a majority of them were involved in hydrolase and binding. Additionally, R. pedestris saliva contained abundant bug-specific proteins with unknown function. Transient expression of salivary proteins in Nicotiana benthamiana leaves identified that RpSP10.3, RpSP13.4, RpSP13.8, RpSP17.8, and RpSP10.2 were capable of inducing cell death, reactive oxygen species (ROS) burst, and hormone signal changes, indicating the potential roles of these proteins in eliciting plant defenses. Our results will shed more light on the molecular mechanisms underlying the plant–insect interactions and are useful for pest management.

Insect cuticular hydrocarbons (CHCs) are organic compounds of the surface lipid layer, which function as a barrier against water loss and xenobiotic penetration, while also serving as chemical signals. Plasticity of CHC profiles can vary depending upon numerous biological and environmental factors. Here, we investigated potential sources of variation in CHC profiles of Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, which are considered to be the most important rice pests in Asia. CHC profiles were quantified by GC/MS, and factors associated with variations were explored by conducting principal component analysis (PCA). Transcriptomes were further compared under different environmental conditions. The results demonstrated that CHC profiles differ among three species and change with different developmental stages, sexes, temperature, humidity and host plants. Genes involved in cuticular lipid biosynthesis pathways are modulated, which might explain why CHC profiles vary among species under different environments. Our study illustrates some biological and ecological variations in modifying CHC profiles, and the underlying molecular regulation mechanisms of the planthoppers in coping with changes of environmental conditions, which is of great importance for identifying potential vulnerabilities relating to pest ecology and developing novel pest management strategies.

For the past 4 decades, intensive molecular studies of mostly leaf mesophyll cell-infecting pathogens and chewing insects have led to compelling models of plant-pathogen and plant-insect interactions. Yet, some of the most devastating pathogens and insect pests live in or feed on the phloem, a systemic tissue belonging to the plant vascular system. Phloem tissues are difficult to study, and phloem-inhabiting pathogens are often impossible to culture, thus limiting our understanding of phloem-insect/pathogen interactions at a molecular level. In this Perspective, we highlight recent literature that reports significant advances in the understanding of phloem interactions with insects and prokaryotic pathogens and attempt to identify critical questions that need attention for future research. It is clear that study of phloem-insect/pathogen interactions represents an exciting frontier of plant science, and influx of new scientific expertise and funding is crucial to achieve faster progress in this important area of research that is integral to global food security.

The brown planthopper, Nilaparvata lugens Stal, has become a major threat in tropical Asian and China since the rice green revolution of the 1960s. Currently, insecticide application remains the primary choice for controlling this rice insect pest, but heavy use of insecticides poses dangerous risks to beneficial natural enemies and pollinators, and stimulates N. lugens reproductivity, and has caused a resurgence of the pest in the major rice-planting regions throughout Asia. Achieving the long-lasting goal of sustainable management of N. lugens requires understanding of the molecular basis of outbreaks of the pest and the development of environment-friendly pest-control strategies. Here, we review the recent molecular advances in N. lugens research on the aspects of its endosymbionts, virus transmission, insecticide resistance, and interaction between N. lugens and rice plants. We also put forward further research directions that may shed some lights on management of the rice pest.

Many insects can live on water and survive being caught in the rain. Current research has shown that insect cuticular hydrocarbons (CHC) confer desiccation resistance to maintain water balance. In this study, we identified a fatty acyl-CoA reductase gene (NlFAR) of the rice brown planthopper, Nilaparvata lugens that is essential for the production of CHCs, and found that NlFAR is essential for N. lugens to walk and jump on water when moving from one rice plant to another in paddy fields. NlFAR was mainly expressed in the integument at the beginning of each molt. Cuticular surface analysis by scanning electron microscopy and characterization of CHC extracts indicated that N. lugens with knockdown of NlFAR using RNA inference (RNAi) had a neater epicuticle layer and a significant decrease in CHC contents. Knockdown of NlFAR did not influence the desiccation resistance of N. lugens, but the dsNlFAR-treated insects were easily adhered and moistened by water droplets or their own secreted honeydew and unable to walk or jump on water. These results suggested that NlFAR is a crucial enzyme for CHC biosynthesis and cuticle waterproofing, but not for water retention of N. lugens, which may provide a potential strategy for pest management.

The Toll pathway plays an important role in defense against infection of various pathogenic microorganisms, including viruses. However, current understanding of Toll pathway was mainly restricted in mammal and some model insects such asDrosophilaand mosquitoes. Whether plant viruses can also activate the Toll signaling pathway in vector insects is still unknown. In this study, using rice stripe virus (RSV) and its insect vector (small brown planthopper,Laodelphax striatellus) as a model, we found that the Toll pathway was activated upon RSV infection. In comparison of viruliferous and non-viruliferous planthoppers, we found that four Toll pathway core genes (Toll,Tube,MyD88, andDorsal) were upregulated in viruliferous planthoppers. When the planthoppers infected with RSV, the expressions ofTollandMyD88were rapidly upregulated at the early stage (1 and 3 days post-infection), whereasDorsalwas upregulated at the late stage (9 days post-infection). Furthermore, induction of Toll pathway was initiated by interaction between a Toll receptor and RSV nucleocapsid protein (NP). Knockdown ofTollincreased the proliferation of RSV in vector insect, and the dsToll-treated insects exhibited higher mortality than that of dsGFP-treated ones. Our results provide the first evidence that the Toll signaling pathway of an insect vector is potentially activated through the direct interaction between Toll receptor and a protein encoded by a plant virus, indicating that Toll immune pathway is an important strategy against plant virus infection in an insect vector.

Persistent plant viruses multiply and circulate inside insect vectors following the route of midgut-hemolymph-salivary gland. Currently, how viruses interact with insect vectors after they are released into hemolymph is not entirely clear. In this study, we found that the hemolymph and fat body (HF) contained the highest Rice stripe virus (RSV) levels. Proteomic analysis on RSV-free and RSV-infected HF identified 156 differentially expressed proteins (DEPs), with the majority of them participating in metabolism, transportation, and detoxification. The RNA binding protein esf2 was the most downregulated protein. Knocking down the expression of esf2 did not influence the RSV burden, but caused the lethal effect to L. striatellus. In contrast, the mRNA decay protein ZFP36L1 was 69% more abundant upon RSV infection, and suppression of ZFP36L1 significantly increased the RSV burden. Our results reveal the potential role of ZFP36L1 in restricting the viral proliferation, and provide valuable clues for unravelling the interaction between RSV and L. striatellus in HF. Significance More than 76% of plant viruses are transmitted by insect vectors. For persistent propagative transmission, plant viruses multiply and circulate inside insects following the route of midgut-hemolymph-salivary gland. However, how viruses interact with vector insects after they are released into hemolymph is not entirely clear. Our study investigated the influence of rice stripe virus (RSV) on insect hemolymph and fat body by iTRAQ labeling method. Among the 156 differentially expressed proteins (DEPs) identified, two proteins associated with mRNA metabolism were selected for function analysis. We found that the mRNA decay activator protein ZFP36L1 influenced the RSV proliferation, and RNA binding protein esf2 caused the lethal effect to L. striatellus. Our results provide valuable clues for unveiling the interaction between RSV and L. striatellus, and might be useful in pest management.

Cucumber green mottle mosaic virus (CGMMV), a critical plant virus, has caused significant economic losses in cucurbit crops worldwide. It has not been proved that CGMMV can be transmitted by an insect vector. In this study, the physical contact transmission of CGMMV by Myzus persicae in Nicotiana benthamiana plants was confirmed under laboratory conditions. The acquisition rate increased with time, and most aphids acquired CGMMV at 72 h of the acquisition access period (AAP). Besides, the acquired CGMMV was retained in the aphids for about 12 h, which was efficiently transmitted back to the healthy N. benthamiana plants. More importantly, further experiments suggested that the transmission was mediated by physical contact rather than the specific interaction between insect vector and plant virus. The results obtained in our study contribute to the development of new control strategies for CGMMV in the field.

Chemosensory proteins (CSP) afford insects the ability to recognize complicated environmental cues and are involved in diverse physiological processes. The planthoppers Laodelphax striatellus and Sogatella furcifera are serious specialist pests of rice throughout eastern Asia. In this study, we identified 21 putative CSP genes (namely LsCSP1–12 and SfCSP1–9) in the two planthoppers L. striatellus and S. furcifera, and profiled their transcript accumulation in different developmental stages and body parts by real-time quantitative PCR (qPCR). All CSPs had the characteristic typical features of the CSP family, with the exception of LsCSP10 and SfCSP2, which lacked the predicted signal peptide sequence. Among these putative CSP genes, only two (LsCSP9 and SfCSP5) were more highly expressed in the antennae, indicating that they might be involved in olfaction. On the contrary, eleven genes (LsCSP2, 3, 4, 6, 8, 12; SfCSP1, 4, 6, 7, 8) and one gene (SfCSP9) were more highly expressed in the legs and heads (without antennae), respectively. The remainders of the genes were ubiquitously expressed in the heads (without antennae) and the bodies (without the legs and heads). The broad expression patterns in different body parts indicate that the CSPs of these two delphacid species may be involved in diverse functions besides chemosensation. These findings increase the understanding of the chemosensory protein system of Hemiptera species.

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.

Significance The cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins encoded in the genome. Why does an insect need so many different CPs? In this study, we use comprehensive large-scale technologies to study the full complement of CPs and their functions in the brown planthopper (BPH). A total of 32 of the 140 BPH CP genes are found to be essential for nymph/adult development, egg production, or embryo development; in addition, redundant and complementary functions of CPs are revealed., Cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins. Why does an insect encode so many different CP genes in the genome? In this study, we use comprehensive large-scale technologies to study the full complement of CPs (i.e., the CP-ome) of the brown planthopper (BPH), Nilaparvata lugens, a major rice plant pest. Eight CP families (CPR, CPF, TWDL, CPLCP, CPG, CPAP1, CPAP3, and CPAPn) including 140 proteins in BPH, in which CPAPn is a CP family that we discovered. The CPG family that was considered to be restricted to the Lepidoptera has also been identified in BPH. As reported here, CPLCP family members are characterized by three conserved sequence motifs. In addition, we identified a testis protein family with a peritrophin A domain that we named TPAP. We authenticated the real existence of 106 proteins among the 140 CPs. RNA interference (RNAi) experiments were conducted against 135 CP genes in early- and late-instar nymphs and newly emerged female adults, demonstrating that 32 CPs were essential for BPH normal development or egg production. Combined RNAi experiments suggested redundant and complementary functions of the large number of CPs. Transcriptomic data revealed that the CP genes were expressed in a tissue-specific manner, and there were four clusters of developmental expression patterns. This study gives a comprehensive understanding of the roles of CPs in an insect cuticle.

Hox genes encode transcriptional regulatory proteins that control axial patterning in all bilaterians. The brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is a destructive insect pest of rice plants in Asian countries. During analysis of the N. lugens transcriptome, we identified a Hox3-like gene (NlHox3) that was highly and specifically expressed in the embryonic stage. We performed functional analysis on the gene to identify its roles in embryonic development and its potential use as a target in RNA interference (RNAi) based pest control. The sequence analysis showed that NlHox3 was homologous to the Hox3 gene and was most closely related with zen of Drosophila. There were no significant differences in oviposition between the treated and control females after injecting double-stranded RNA of NlHox3 (dsNlHox3) into newly emerged female adult BPHs; however, there was a significant difference in the hatchability of those eggs laid, which no egg from the treated group hatched normally. Injecting female adult BPHs with dsNlHox3 led to necrosis of these offspring embryos, with eye reversal and undeveloped organs, suggesting that NlHox3 was an essential gene for embryonic development and might be a potential target for RNAi-based control of this insect pest.

A number of sap-sucking insects harbor endosymbionts, which are thought to play an important role in the development of their hosts. One of the most important rice pests, the brown planthopper (BPH), Nilaparvata lugens (Stål), harbors an obligatory yeast-like symbiont (YLS) that cannot be cultured in vitro. Genomic information on this YLS would be useful to better understand its evolution. In this study, we performed genome sequencing of the YLS using both 454 and Illumina approaches, generating a draft genome that shows a slightly smaller genome size and relatively higher GC content than most ascomycete fungi. A phylogenomic analysis of the YLS supported its close relationship with insect pathogens. We analyzed YLS-specific genes and the categories of genes that are likely to have changed in the YLS during its evolution. The loss of mating type locus demonstrated in the YLS sheds light on the evolution of eukaryotic symbionts. This information about the YLS genome provides a helpful guide for further understanding endosymbiotic associations in hemiptera and the symbiotic replacement of ancient bacteria with a multifunctional YLS seems to have been a successful change.

β-N-Acetylhexosaminidases (HEXs) are enzymes that can degrade the chitin oligosaccharides that are produced by the activity of chitinases on chitin in insects. Using bioinformatic methods based on genome and transcriptome databases, 11 β-N-acetylhexosaminidase genes (NlHexs) in Nilaparvata lugens were identified and characterized. Phylogenetic analysis revealed a six-grouped tree topology. The O-Linked N-acetylglucosaminidase (O-GlcNAcase) group includes NlHex11, which harbours a catalytic domain that differs from that of the other 10 NlHexs. Observations of the expression of NlHexs during different developmental stages revealed that NlHex4 is expressed with periodicity during moulting. Although the tissue-specific expression patterns of most NlHexs were nonspecific, NlHex4 was found to be expressed mainly in the female reproductive system as well as in the integument. RNA interference (RNAi) demonstrated failure to shed the old cuticle only in the nymphs treated with double-stranded RNA (dsRNA) targeting NlHex4, and these nymphs eventually died; no observable morphological abnormalities were found in insects treated with dsRNAs targeting the other 10 NlHexs. Based on this study and our previous analyses, a '5 + 1 + 3' pattern of chitinolytic enzymes is proposed, in which five chitinases, one NlHEX and three chitin deacetylases are required for moulting in N. lugens. A better understanding of chitin metabolism in the hemimetabolous insect, N. lugens, would be achieved by considering three chitinolytic enzyme families: chitinase, chitin deacetylase and β-N-acetylhexosaminidase.

Bicaudal-C (Bic-C) was originally identified in a Drosophila melanogaster mutagenesis screen and plays vital roles in embryogenesis. In this study, we characterized the Bic-C gene in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), an insect pest that undergoes incomplete metamorphosis. Our result showed that N. lugens Bic-C (NlBic-C) is a female-specific gene in this species. It is specifically expressed in developing oocytes and is not expressed in laid eggs. Ribonucleic acid interference (RNAi) of NlBic-C arrested the uptake of vitelline by oocytes, and resulted in undeveloped ovaries and the complete inhibition of oocyte growth in the ovarioles, suggesting that NlBic-C is required for oogenesis and oocyte maturation. NlBic-C is extremely highly sensitive to RNAi, suggesting that it may be a potential target in RNAi-based insect pest management.

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.

In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed.

NlFoxL2 was aligned with FoxL2 orthologs from 16 other insect species using the ClustalX program.

Chitin deacetylases (CDAs) are enzymes required for one of the pathways of chitin degradation, in which chitosan is produced by the deacetylation of chitin. Bioinformatic investigations with genomic and transcriptomic databases identified four genes encoding CDAs in Nilaparvata lugens (NlCDAs). Phylogenetic analysis showed that insect CDAs were clustered into five major groups. Group I, III and IV CDAs are found in all insect species, whereas the pupa-specific group II and gut-specific group V CDAs are not found in the plant-sap/blood-sucking hemimetabolous species from Hemiptera and Anoplura. The developmental and tissue-specific expression patterns of four NlCDAs revealed that NlCDA3 was a gut-specific CDA, with high expression at all developmental stages; NlCDA1, NlCDA2 and NlCDA4 were highly expressed in the integument and peaked periodically during every moulting, which suggests their roles in chitin turnover of the insect old cuticle. Lethal phenotypes of cuticle shedding failure and high mortality after the injection of double-stranded RNAs (dsRNAs) for NlCDA1, NlCDA2 and NlCDA4 provide further evidence for their functions associated with moulting. No observable morphological and internal structural abnormality was obtained in insects treated with dsRNA for gut-specific NlCDA3.

Planthoppers are highly destructive pests that damage rice plants by feeding and transmitting viruses. They feed on phloem sap using specialized mouthparts and secrete saliva during feeding. Over the past decade, genomic, transcriptomic, and proteomic approaches have greatly improved our understanding of the complexity of planthopper saliva, and have provided a glimpse of planthopper-plant interactions. Here we focus on a few recent advances in planthopper saliva and discuss how salivary components influence planthopper performance. Understanding the molecular basis of saliva in planthopper-plant interactions will provide evolutionary insights, and promote the development of novel strategies for controlling agricultural pests.

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.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects the domestic silkworm. In this study, six BmNPV strains were compared at the whole genome level. We found that the number of bro genes and the composition of the homologous regions (hrs) are the two primary areas of divergence within these genomes. When we compared the ORFs of these BmNPV variants, we noticed a high degree of sequence divergence in the ORFs that are not baculovirus core genes. This result is consistent with the results derived from phylogenetic trees and evolutionary pressure analyses of these ORFs, indicating that ORFs that are not core genes likely play important roles in the evolution of BmNPV strains. The evolutionary relationships of these BmNPV strains might be explained by their geographic origins or those of their hosts. In addition, the total number of hr palindromes seems to affect viral DNA replication in Bm5 cells.

article i nfo A Bombyx mori continuous cell line, designated DZNU-Bm-17, was established from larval ovaries. The cells were initially grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum and 3% heat inactivated B. mori hemolymph at 25±1 °C and later adapted gradually to TNM-FH medium. Partially adhered refractive cells were the predominant cell type in the culture. The cells took about 1055 days to complete 100 passages in TNM-FH medium. The population doubling time of the cell line was about 30-34 h at 25±1 °C. The cell population was largely diploid, but a few triploids and tetraploids were also observed. DNA profiles using simple sequence repeat loci established the differences between the DZNU-Bm-1, Bm-5, DZNU-Bm-12, DZNU-Bm-17, and BmN cell lines. The cell line was susceptible to budded virus of B. mori nucleopolyhedrovirus (BmNPV), and 85-92% of the cells harbored BmNPV with an average of 15 occlusion bodies/infected cell. The cells expressed the luciferase and green fluorescent proteins using the BmNPV bacmid vector.We suggest the usefulness of the DZNU-Bm-17 cell line for BmNPV-based baculoviral ex- pression studies.

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’.

Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes, and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath, while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests.

The brown planthopper, Nilaparvata lugens (N. lugens, Hemiptera: Delphacidae), and the green rice leafhopper, Nephotettix cincticeps (N. cincticeps, Hemiptera: Cicadellidae), two sap-sucking feeders, have caused many destructive agricultural disasters in East Asia, as they can bring diseases like 'hopper burn' and transmit plant viruses. Recently, continuously-cultured cell lines from both insects have been reported. However, exogenous protein expression systems have not yet been established. Here, we identified thirteen tubulin genes and three actin genes from N. lugens, and one tubulin gene and two actin genes from N. cincticeps. Furthermore, putative promoter regions of these genes were analyzed by bioinformatic approaches and 5'-RACE assay, and the promoter strength was evaluated by driving the enhanced green fluorescent protein expression in three insect cell lines, S2, Sf9, and BmN. Finally, we identified three effective promoters (Nl_αTub1 promoter, Nl_act3 promoter, and Nc_act1 promoter) among all candidates we screened. The Nc_act1 promoter showed the strongest activity, while the Nl_αTub1 promoter only worked in S2 cells. In conclusion, we identified and functionally characterized three native promoters from N. lugens and N.cincticeps, which would facilitate the establishment of exogenous protein expression systems suitable for these two insect pests.

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.

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects domestic silkworm. BmNPV ORF71 (Bm71) is not a core set gene in baculovirus and shares 92 % amino acid sequence identity with Autographa californica multinucleocapsid NPV ORF88 (Ac88/cg30). Previously, it has been reported that virus lacking Ac88 had no striking phenotypes in cell lines or host larvae. However, the exact role of Bm71 during BmNPV life cycle remains unknown. In the present study, we constructed a Bm71-disrupted (Bm71-D) virus and assessed the effect of the Bm71 disruption on viral replication and viral phenotype throughout the viral life cycle. Results showed that the Bm71-D bacmid could successfully transfect Bm5 cell lines and produce infectious budded virus (BV). But the BV titer was 10- to 100-fold lower than that of the wild-type (WT) virus during infection, and the decreased BV titer was rescued by Bm71 gene repair virus (Bm71-R). A larval bioassay showed that Bm71-D virus took 7.5 h longer than the WT to kill Bombyx mori larvae. Transmission electron microscopy analysis indicated that the Bm71-D virus-infected cells had typical virogenic stroma, bundles of nucleocapsids and polyhedra. Taken together, these results suggest that Bm71 has important implications for determining BV yield and virulence in viral life cycle even though it is not an essential gene for replication of BmNPV.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). Because AChE is expressed in all invertebrate and vertebrate animals as a key enzyme of the cholinergic system, the toxicity of anticholinesterase insecticides to mammals and non-target species such as beneficial insects has been a great concern. In addition, the intensive use of OP and CB insecticides has resulted in the development of resistance in many insect pests, which has limited the use of anticholinesterase insecticides. Many aces encoding AChEs have been sequenced from a variety of vertebrates, insects and other invertebrates, and crystal structures of four AChEs have been determined in the past 20 years. Although the primary motifs and the three dimensional (3D) structures of different AChEs are similar, differences among AChEs are obvious. The catalytic properties and inhibition kinetics of AChEs from different groups of insects and mammals may be quite different, and two AChEs from a single insect may also show distinct differences. These differences may provide new opportunities for designing more selective insecticides for pest management.

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.

The begomoviruses are the largest and most economically important group of plant viruses transmitted exclusively by the whitefly Bemisia tabaci in a circulative, persistent manner. The circulation of the viruses within the insect vectors involves complex interactions between virus and vector components; however, the molecular mechanisms of these interactions remain largely unknown. Here we investigated the transcriptional response of the invasive B. tabaci Middle East-Asia Minor 1 species to Tomato yellow leaf curl China virus (TYLCCNV) using Illumina sequencing technology. Results showed that 1,606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that TYLCCNV can perturb the cell cycle and primary metabolism in the whitefly, which explains the negative effect of this virus on the longevity and fecundity of B. tabaci . Our data also demonstrated that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the viruliferous whitefly. Furthermore, PCR results showed that TYLCCNV can invade the ovary and fat body tissues of the whitefly, and Lysotracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both the ovary and fat body tissues. Surprisingly, TYLCCNV also suppressed the whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways. Taken together, these results reveal the relationship of coevolved adaptations between begomoviruses and whiteflies and will provide a road map for future investigations into the complex interactions between plant viruses and their insect vectors.

Acetylcholinesterase (AChE) is the primary target of organophosphate (OP) and carbamate (CB) insecticides. Many insect species have been shown to have two different AChE genes. The amino acid identity between the two lepidopteran AChEs is lower than 40%, and potential differences in enzymatic function have not been characterized. In this study, the cDNAs encoding two AChEs (Boma-AChE1 and Boma-AChE2) from Bombyx mandarina were sequenced, and the corresponding proteins were heterologously expressed to compare their enzymatic properties and interactions with insecticides in vitro. Both of these enzymes had high specific activities for acetylthiocholine iodide. Studies on substrate and inhibitor specificities confirmed that both enzymes belong to AChE. Insecticide inhibition assays indicated that Boma-AChE1 was more sensitive than Boma-AChE2 to eight of the nine insecticides tested. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos. The results suggested that two AChEs from a lepidopteran insect have distinct catalytic properties and responses to different inhibitors.

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus- insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I(50) values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.

BM47 is encoded by open reading frame 47 of Bombyx mori nucleopolyhedrovirus (BmNPV). BM47 was localized in the nucleus of BmNPV-infected cells. In the present study, we investigated a novel nuclear localization signal (NLS) for BM47 transport and accumulation in the nucleus. By expressing various regions of BM47 fused to enhanced green fluorescent protein (EGFP), we demonstrated that residues 117-148 are necessary for mediating nuclear localization of BM47. Site-directed mutation analysis showed that the two basic residue clusters at positions 117-120 (¹¹⁷RKRR) and 144-148 (¹⁴⁴RKR-K) constitute an authentic NLS for BM47 localization. Finally, we observed that two clusters of basic residues were conserved in BM47 homologues of group-I nucleopolyhedroviruses.

Open reading frame 60 (bm60) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a conserved gene among group I and some group II NPVs. bm60 encodes a late expressed protein that localizes to both the cytoplasm and nucleus of infected cells. This paper describes the characterization of a BmNPV mutant (vbm60-Null) lacking functional bm60. It was observed that the production of budded virus (BV) was reduced by nearly an order of magnitude relative to wt virus in vbm60-Null-infected BmN cells and B. mori larvae. Quantitative real-time PCR assay showed that the viral DNA replication was affected in infected cells due to disruption of bm60. Larval bioassays showed that the speed of kill of vbm60-Null virus was greatly reduced, as it took approximately 28-36 h longer to kill the fifth instar B. mori larvae. These results suggest that BmNPV bm60 is not essential for viral replication, but required for efficient BV production.

The infection profiles of the Bombyx mori nucleopolyhedrovirus (BmNPV) in B. mori larvae revealed that the virus invaded the fat body and haemocyte of both KN and 306 strains, which are highly resistant and susceptible, respectively, to BmNPV infection. However, viral proliferation was notably slowed in the resistant B. mori strain. Using suppression subtractive hybridization, two fat body cDNA libraries were constructed to compare BmNPV responsive gene expression levels between the two silkworm lines. In total, 96 differentially expressed genes were obtained. Real-time quantitative PCR (qPCR) analysis confirmed that eight genes were significantly up-regulated in the fat body and haemocyte of the KN strain following BmNPV injection. Our results suggest that these genes may have potential roles in B. mori antiviral infection mechanisms.

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.

A new continuous cell line from ovarian tissue of commercial variety "Kolar Gold" of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 +/- 1 degrees C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85-90% of the cells harboring BmNPV and having an average of 3-17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm(De)) and a repaired virus (vBm(Re)) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD(50) bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT(50) bioassay.

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.

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

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.

The open reading frame 122 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm122) has been observed to be a conserved gene in the lepidopteran baculoviruses that have been completely sequenced so far. Its transcript was detected at 3 h post infection (h p.i.) and remained detectable at up to 96 h p.i. Temporal transcription analysis indicated that Bm122 is transcribed by host RNA polymerase. The size of the translational product of the Bm122 gene in Tn5B-1-4 cells was approximately 23 kDa, which is in agreement with the predicted value of 22.9 kDa, suggesting that no major posttranslational modification occurred in the primary protein product. The subcellular localization of Bm122 was studied using EGFP-Bm122, which revealed that Bm122 protein was accumulated within the nuclear region of virus-infected BmN cells. All these results suggest that Bm122 is an early gene encoding a protein that functions in the nucleus.

Complementary DNAs encoding two types of acetylcholinesterase (AChE) were isolated from the silkworm, Bombyx mori. The type 1 (Bmace1) and type 2 (Bmace2) ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and C-terminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni (Tn-5B1-4) cells. The resulting products exhibited AChE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the ace1-type enzyme to inhibition by eserine and paraoxon.

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.

The complete nucleotide sequence of Ectropis obliqua nucleopolyhedrovirus (EcobNPV), which infects the tea looper caterpillar, was determined and analyzed. The double stranded circular genome is composed of 131,204 bp and is 37.6% G+C rich. The analysis predicted 126 putative, minimally overlapping open reading frames (ORFs) with 150 or more nucleotides that together compose 89.8% of the genome. The remaining 10.2% constitute non-coding and three homologous regions. Comparison with previously sequenced baculoviruses indicated that three ORFs were unique to EcobNPV, while the remaining 123 ORFs shared identity with other baculovirus genes. In addition to two bro homologues, three other repeat ORFs, including dbp, p26, and odv-e66, were identified. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EcobNPV is a Group II NPV, although its genomic organization was highly distinct.

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.