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.Keywords: baculovirus; Betabaculovirus; ODV proteome; Pieris rapae granulovirus (PrGV);

Diatoms constitute a remarkably diverse and attractive group of microalgae, serving as the main primary producers in many ecosystems and a potential source of renewable biofuel. The enhancement of lipid production in diatoms has been achieved by the optimization of culture conditions, such as temperature, salinity, and nutrient starvation. In this study, we performed Illumina sequencing and the de novo transcriptome assembly of an oleaginous diatom, Nitzschia sp., which produces up to 50 % oil by weight under defined conditions. High-quality reads were assembled into 28,117 isogenes and then subjected to BLAST alignment, Gene Ontology annotation, and KEGG Orthology annotation. The majority of genes and pathways related to cell wall formation and lipid biosynthesis were identified by these analyses. In addition, elevated salinity was found to increase the total lipid content of Nitzschia sp. For a better understanding of the molecular mechanisms regulating this phenomenon, transcriptome profiles under different conditions of salinity were compared to examine how the metabolic flux was channeled to increase the biosynthesis of triacylglycerols. As expected, a subset of genes involved in lipid biosynthesis was up-regulated under salinity stress. Meanwhile, carbon and nitrogen metabolism genes were also significantly affected, indicating a diversion of metabolic pathways. The data we generated here enrich the genomic resources available for non-model algae and provide insights into the mechanisms of lipid accumulation in microalgae.

The brown planthopper Nilaparvata lugens harbors intracellular fungal yeast-like symbionts and endosymbiotic bacteria, with the latter mainly comprising Wolbachia and Arsenophonus. In this study, Wolbachia or Arsenophonus were detected in all 15 brown planthopper populations collected from China and Southeastern Asian countries. Furthermore, Polymerase Chain Reaction (PCR) analysis of the individuals in a population that was infected by both Wolbachia and Arsenophonus showed that each individual was infected by only one of the two symbiotic bacteria. Real-time quantitative PCR showed that both endosymbionts are mainly localized in the mycetocytes of the fat body. Reciprocal crosses between the Wolbachia+ and Arsenophonus+ brown planthopper populations showed that both bacteria were maternally transmitted. Our results showed that the brown planthopper populations are extensively infected by Wolbachia or Arsenophonus, and the two bacteria may be exclusive in each host individual. This finding might be helpful for further studies on the biological functions of the endosymbiotic bacteria and will deepen our understanding of the complicated symbiosis system in this host.

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.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat/hydA). The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic fermentation for the production of hydrogen from glucose was investigated using E. aerogenes ATCC13408 and the recombinant strain. The results showed that the hydrogen yield markedly increased, from 442.82 ± 22.61 ml/g glucose in the ATCC13408 strain to 864.02 ± 36.8 ml/g glucose in the recombinant. The maximum rate of hydrogen production was found to be 53.49 ± 3.34 ml l−1 h−1 using 1% (w/v) glucose as the substrate at pH 6.0 and a reaction temperature of 37°C.

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.

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.

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.

Euproctis pseudoconspersa NPV (EupsNPV) is pathogenic to the tea tussock (E. pseudoconspersa), one of the major pests of tea bushes in East Asia, and has been used to control the pest. Electron microscope observation showed there were two modes for the virions embedded in each polyhedron, single-nucleocapsid and double-nucleocapsid. The EupsNPV genome contained 141,291 bp and had a G + C content of 40.4%. Of 139 potential ORFs predicted from the sequence, 126 had a homology in other baculoviruses; 13 were unique to EupsNPV. Four homologous repeat sequences (hrs) were present in the EupsNPV genome and the repeat sequences were different between these hrs. Three ORFs were identified to contain two homologues in the EupsNPV genome, including bro, p26 and dbp. Gene parity plots, percent identities of gene homologues and phylogenetic analysis all suggested that EupsNPV is most closely related to EcobNPV in Group II NPV, although its genomic organization was highly distinct.

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC50) and lethal time (LT50) were conducted to test the susceptibility of I. fasciata larvae to the virus.

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.

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.Graphical abstractDownload high-res image (190KB)Download full-size imageHighlights► Most insects have both CHS1 and CHS2, but sap-sucking N. lugens possesses only CHS1. ► CHS1 from N. lugens and other sequenced insects but Acyrthosiphon pisum, have two transcript variants. ► CHS1a and CHS1b had different tissue-specific and developmental expression patterns. ► RNAi-mediated gene silencing of CHS1 or CHS1a led to lethal phenotype. ► Silencing CHS1 resulted in elongated wing pads and “wasp-waisted” phenotype.

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.Download high-res image (69KB)Download full-size imageHighlights► Two reeler domain-containing cDNA clones, reeler1 and reeler2 were isolated from Bombyx mori. ► Reeler1 expression is inducible by bacterial challenge in B. mori larvae, while reeler2 expression is not inducible. ► Reeler1 is involved in the nodulation responses. ► Reeler1 participates in the prophenoloxidase activation cascade.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

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.

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.

•Native structure of MCMV is reconstructed by single particle analysis.•The C-terminus resolvability reveals new contributions to the stability of MCMV.•Negative charges were found in the centers of the pentamers and hexamers.•The most exposed loops that potentially affect MCMV transmission are indicated.Maize chlorotic mottle virus (MCMV) is the only member of the Machlomovirus genus in the family Tombusviridae. Here, we obtained the Cryo-EM structure of MCMV by single particle analysis with most local resolution at approximately 4 Å. The Cα backbone was built based on residues with bulky side chains. The resolved C-terminus of the capsid protein subunit and obvious openings at the 2-fold axis demonstrated the compactness of the asymmetric unit, which indicates an important role in the stability of MCMV. The Asp116 residue from each subunit around the 5-fold and 3-fold axes contributed to the negative charges in the centers of the pentamers and hexamers, which might serve as a solid barrier against the leakage of genomic RNA. Finally, the loops most exposed on the surface were analyzed and are proposed to be potential functional sites related to MCMV transmission.Download high-res image (610KB)Download full-size image

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 (Stål) (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.Graphical abstractDownload high-res image (223KB)Download full-size imageHighlights► Flightin is conserved in Pancrustacea, which includes hexapods and crustaceans. ► Flightin was found in the tymbal muscles of the planthopper male adult. ► Flightin was found in the indirect flight muscles of the long-winged planthopper adult. ► Flightin was not detected in the thorax of the short-winged planthopper adult. ► Flightin localized to Daphnia magna antenna muscles.

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.Download high-res image (190KB)Download full-size imageHighlights► DSC1 orthologs were found in all insect genomes and in a few other invertebrates, but not in any vertebrates. ► Two full-length DSC1 orthologs were isolated from Nilaparvata lugens and Bombyx mori. ► DSC1 orthologs share unique structural features. ► SC1s form a single family of voltage-gated cation channels distinct from classical Ca2+ channels or Na+ channels.

Acetylcholinesterase (AChE), which is encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. AChE is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). In this study, full-length cDNA sequences of two ace genes (Nlace1 and Nlace2) were sequenced from the brown planthopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1 cDNA is 2842 nucleotides long and contains an ORF potentially encoding a 790 amino acid peptide. Nlace2 cDNA is 2852 bp in length and contains an ORF that potentially encodes a 672 amino acid peptide. NlAChE1 has an identity of 40% with NlAChE2 at the amino acid sequence level. Phylogenetic analysis of 59 AChEs from 32 animal species showed that NlAChE1 is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while NlAChE2 is most closely related to AChE2 from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1 is expressed at a much higher level than Nlace2 in all developmental stages and tissues, demonstrating that NlAChE1 may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.Graphical abstractDownload full-size imageHighlights► Full-length cDNA’s of two acetylcholinesterase genes were sequenced from Nilaparvata lugens. ► Nlace1 is expressed at a much higher level than Nlace2. ► Nlace1 is most closely related to ace1 from Blattella, not those from the same order.

•Six BmNPV strains were compared based on whole genomic sequences.•Evolutionary pressure of each viral ORF was analyzed.•bro genes and the composition of homologous regions (hrs) are two primary areas of divergence.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.

The voltage-gated sodium channel mediates the rapid rising phase of action potentials in almost all excitable cells and is a molecular target of a variety of neurotoxins including pyrethroid insecticides. Most studies have focused on the expression of sodium channel genes in the adult stage, information on other developmental stages, however, is limited. In this study, we characterized the para sodium channel orthologous gene (BmNav) of the silkworm Bombyx mori, a model insect of Lepidopteran species. The BmNav gene covers a 31 kb genome region and contains 36 exons. The longest ORF contained 6258 bp and encoded 2085 amino acid residues, which shares 74%, and 77% overall amino acid sequence identities with the sodium channel proteins from Drosophila melanogaster and Blattella germanica, respectively. Using high-throughput Solexa sequence technology we conducted sequence analysis of BmNav cDNAs from embryos, larvae, pupae and adults of the silkworm, identified alternative splicing sites and determined the frequencies of these splicing events in four developmental stages. Three optional exons, two sets of mutually exclusive exons, and one internal spliced exon were identified. One optional exon is unique to BmNav, while the others are conserved in other insect sodium channel genes. Interestingly, the expression of the mutually exclusive exons is developmentally regulated.

A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.

•First characterization of Bicaudal-C gene in a non-Drosophila insect.•NlBic-C is specifically expressed in developing oocytes but not in laid eggs.•NlBic-C is required for oogenesis and oocyte maturation.•The gene is extremely highly sensitive to the RNA interference.•This gene is a potential target in RNAi-based planthopper management.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.Graphical abstractDownload full-size image

We investigated variations in the gene expression of Bombyx mori following infection with a nucleopolyhedrovirus (BmNPV). Two B. mori strains, KN and 306, which are highly resistant and susceptible to BmNPV infection, respectively, were used in this study. The infection profiles of BmNPV in the B. mori KN and 306 larvae revealed that the virus invaded the midguts of both these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, two cDNA libraries were constructed in order to compare the BmNPV responsive gene expressions between the two silkworm lines. In total, 62 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that eight genes were significantly up-regulated in the midgut of the KN strain following BmNPV infection. Our results imply that these up-regulated genes may be involved in the B. mori immune response against BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF118 (bm118) is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF142, one of the core genes existing in all baculovirus genomes sequenced to date, suggesting that Bm118 plays a critical role in viral infection. In this study, the primary role of Bm118 was investigated by using homologous recombination in Escherichia coli to generate a bm118 knockout bacmid containing the BmNPV genome. In addition, the bm118 rescue bacmid was constructed by transposing a bm118 gene cassette into the polh locus of the bm118 knockout bacmid. Transfection assays demonstrated that the bm118 knockout bacmid was incapable of producing budded virion (BV). Nevertheless, this defect could be partially recovered by a rescue bacmid. Electron microscopy analysis revealed that the bm118 knockout produced aberrant capsids characterized by translucent, elongated nucleocapsids present as bundles within the nuclei. This construct also produced polyhedra lacking virions. These results reveal that Bm118 is essential for BV production and nucelocapsid maturation.

•NlMul, a salivary sheath and gel protein, essential for normal sheath formation.•NlMul expression was enhanced when feeding on the resistant rice variety.•NlMul is essential for brown planthopper feeding on the resistant rice plant.•First gene confirmed to be related to planthopper virulence and host adaptation.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.Download high-res image (128KB)Download full-size image

•TOR pathway controls accessory gland and deferent duct development.•Knockdown Tor gene resulted delivery failure of sperm from male to female.•Knockdown Tor gene resulted male infertility.•TOR pathway also controls male copulatory organ development.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.Download high-res image (83KB)Download full-size image

The brown planthopper (Nilaparvata lugens, BPH) is the most serious rice plant pests in Asia. In this study, we performed transcriptome-wide analysis on BPH intestine. We obtained more than 26 million sequencing reads that were then assembled into 53,553 unigenes with a mean size of 388 bp. Based on similarity search with the nucleotide sequences available at NCBI, BPH intestine-specific transcriptome analysis identified 21,405 sequences. Assembled sequences were annotated with gene description, gene ontology and clusters of orthologous group terms. The digestion-, defense- and xenobiotic metabolism-related genes were abundantly detected in the transcripts from BPH intestine. Many novel genes including 33 digestion-related genes, 25 immune responsive genes and 27 detoxification-related genes are first reported here. We investigated the gene expression patterns at the transcript levels in different tissues by quantitative real-time PCR analysis, which revealed that some genes had intestine-specific expression, implicating their potential significance for BPH management.Highlights► De novo intestine-specific transcriptome of the brown planthopper. ► Digestion-, defense- and xenobiotic metabolism-related genes abundantly detected. ► Intestine-specific expression genes identified. ► Potential targets for BPH control using RNAi-based transgenic rice.

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.

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 (vBmDe) and a repaired virus (vBmRe) 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 LD50 bioassay and took 18–23 h longer to kill larvae than wild-type virus in an LT50 bioassay.