Qili Feng

Sequence and Organization of the Neodiprion lecontei Nucleopolyhedrovirus Genome

ABSTRACT All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.

Structure and expression of glutathione S-transferase genes from the midgut of the Common cutworm, Spodoptera litura (Noctuidae) and their response to xenobiotic compounds and bacteria

Glutathione S-transferases (GSTs) play a pivotal role in detoxifying endogenous and xenobiotic compounds and oxidative stress resistance in cells. In this study, five GST genes, including three Sigma GSTs (SlGSTs1, SlGSTs2, and SlGSTs3), one Omega GST (SlGSTo1) and one un-classified GST (SlGSTu1) were identified from the midgut of the Common cutworm, Spodoptera litura. Structure analyses of the eight (including the previously identified Epsilon GST genes, SlGSTe1, SlGSTe2 and SlGSTe3 from the same insect) SlGSTs genes showed that the Epsilon SlGSTe genes do not contain any intron, while the Sigma SlGSTs contain three introns and the Omega SlGSTo1 and the un-classified SlGSTu1 contain five introns. Analysis of the spatial and temporal expression of these eight SlGSTs indicated that SlGSTe1, SlGSTs2 and SlGSTo1 expressed in all stages of development from the egg to the adult stages. SlGSTe2, SlGSTe3, SlGSTs1, SlGSTs3 and SlGSTu1 had higher expression levels in the larval stages than in other stages and their expression levels in the midgut were higher than in other tissues. SlGSTs1 was expressed in the larval midgut but not in the fat body and could be induced by bacterial infections. The expression of SlGSTe1, SlGSTe3, SlGSTs1 and SlGSTs3 was increased by chlorpyrifos to various degrees, while the expression of SlGSTe1, SlGSTe3, SlGSTs1, SlGSTs3 and SlGSTo1 was increased by xanthotoxin. Levels of malonaldehyde, an indicator of oxidative stress, were higher in the larval midgut than in the pupal midgut. Chlorpyrifos induced the malonaldehyde content in the larvae, whereas xanthotoxin did not. It is hypothesized that high expression levels of the midgut SlGSTs might be due to the increased levels of oxidative stress caused by feeding, bacterial infection and xenobiotic compounds.

Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura

Small heat shock proteins (sHsps) are probably the most diverse in structure and function among the various superfamilies of stress proteins. To explore the diverse functions of insect sHsps, six sHsp cDNAs were cloned from the midgut cDNA library of Spodoptera litura, and a phylogenetic tree was constructed based on the conserved α-crystalline domains. The expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) induction, were studied by real-time quantitative PCR. Based on sequence characteristics and phylogenetic relationships, the six SlHsps were classified into three independent groups: BmHsp20.4 like proteins (SlHsp19.7, 20.4, 20.7, 20.8), BmHsp26.6 like protein (SlHsp20), and BmHsp21.4 like protein (SlHsp21.4). All the SlHsps showed highest expression in the Malpighian tubules. The four BmHsp20.4 like protein genes were up-regulated by thermal stress and showed expression variation with development. SlHsp20 exhibited lower expression levels in both egg and larval stages than in pupal and adult stages. SlHsp21.4 retained a constant expression level during all life stages. The expression of both SlHsp20.4 and SlHsp20.8 was significantly up-regulated by 20E. These results indicate that sHsps play diverse functions in S. litura: the BmHsp20.4 like proteins are involved in both thermal adaptation and development; SlHsp20 does not respond to temperature stress but possibly plays a role in metamorphosis; SlHsp21.4 may have no direct relationship with either thermal response or development.

Homeodomain POU and Abd-A proteins regulate the transcription of pupal genes during metamorphosis of the silkworm, Bombyx mori

A cascade of 20-hydroxyecdysone–mediated gene expression and repression initiates larva-to-pupa metamorphosis. We recently showed that two transcription factors, BmPOUM2 and BmβFTZ-F1, bind to the cis-regulatory elements in the promoter of the gene coding for cuticle protein, BmWCP4, and regulate its expression during Bombyx mori metamorphosis. Here we show that down-regulation of BmPOUM2 expression by RNA interference during the wandering stage resulted in failure to complete metamorphosis. The thorax epidermis of RNA interference-treated larvae became transparent, wing disc growth and differentiation were arrested, and the larvae failed to spin cocoons. Quantitative real-time PCR analysis showed that expression of the genes coding for pupal-specific wing cuticle proteins BmWCP1, BmWCP2, BmWCP3, BmWCP4, BmWCP5, BmWCP6, BmWCP8, and BmWCP9 were down-regulated in BmPOUM2 dsRNA-treated animals, whereas overexpression of BmPOUM2 protein increased the expression of BmWCP4, BmWCP5, BmWCP6, BmWCP7, and BmWCP8. Pull-down assays, far-Western blot, and electrophoretic mobility shift assay showed that the BmPOUM2 protein interacted with another homeodomain transcription factor, BmAbd-A, to induce the expression of BmWCP4. Immunohistochemical localization of BmPOUM2, BmAbd-A, and BmWCP4 proteins revealed that BmAbd-A and BmPOUM2 proteins are colocalized in the wing disc cell nuclei, whereas BmWCP4 protein is localized in the cytoplasm. Together these data suggest that BmPOUM2 interacts with the homeodomain transcription factor BmAbd-A and regulates the expression of BmWCP4 and probably other BmWCPs to complete the larva-to-pupa transformation. Although homeodomain proteins are known to regulate embryonic development, this study showed that these proteins also regulate metamorphosis.

Two epsilon glutathione S-transferase cDNAs from the common cutworm, Spodoptera litura: Characterization and developmental and induced expression by insecticides

Two Spodoptera litura glutathione S-transferase cDNAs (Slgste2 and Slgste3) which were cloned from a midgut cDNA, encoded two structurally distinct proteins with a predicted molecular mass of 25 and 24kDa, respectively. Slgste2 and Slgste3 were single-copy genes in the S. litura genome and there was no intron within the genes. The transcripts and proteins of Slgste2 and Slgste3 were predominately present in the midgut of the 5th and 6th instar larvae. The apparent Vmax of the purified SlGSTE2 and SlGSTE3 recombinant proteins towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. Slgste2 expression was up-regulated by 1-naphthyl methylcarbamate (carbaryl), 1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane (DDT), deltamethrin, tebufenozide (RH5992) and Bacillus thuringiensis (Bt), but not affected by malathion, while Slgste3 expression was slightly up-regulated by carbaryl, Bt and DDT, but not affected by RH5992, malathion and deltamethrin. The results suggest that Slgste2 and Slgste3 may play roles in detoxifying various insecticides in S. litura.

Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura.

Insect glutathione S‐transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up‐regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had Vmax (reaction rate of the enzyme saturated with the substrate) and Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and Vmax and Km values of 22.96 ± 0.78 μmol/min/mg and 0.83 ± 0.106 mmol/L for 1‐chloro‐2,4‐dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro‐diphenyl‐trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.

Protein Profiles of the Midgut of Spodoptera litura Larvae at the Sixth Instar Feeding Stage by Shotgun ESI−MS Approach

By using shotgun HPLC-ESI-MS proteomics approach, 2043 peptides were identified from the midgut of Spodoptera litura larvae at the sixth instar feeding stage, out of which 1489 (72.9%) were found to have their homologues in the public protein databases and 842 had identities of molecular functions. Seven-hundred forty-one peptides were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization, with 336 and 251 peptides being related to catalytic activity and binding activity, respectively. Most of the catalytic proteins had activity of hydrolases, oxidoreductases and transferases and most of the binding proteins were involved in protein-binding activity. Among the annotated peptides, 487 were classified into different cellular processes and 490 were classified to locate in the cytoplasm. Nonredundant enzymes associated with the metabolisms of carbohydrates, lipids and fatty acids, amino acids and proteins, translation, transport, and stress resistance were identified. Presence and expression at high levels of numerous enzymes of glycolysis pathway, synthesis of proteins, and absorption and transport of fatty acids and lipids indicate that active metabolism processes of carbohydrates, proteins, and lipids occurred in the midgut of sixth instar feeding larvae of S. litura. The protein profile provides a basis for further study of the physiological events in the midgut of S. litura.

A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization

A full-length cDNA (Slctlp2) encoding a chymotrypsin-like serine protease was cloned from Spodoptera litura. This cDNA encoded a putative serine protease with a predicted molecular mass of 30.6kDa, which contained a serine protease catalytic motif GDSGGPL. Temporal and spatial expression of Slctlp2 mRNA and protein detected by Northern blotting, RT-PCR, qPCR and Western blotting analyses revealed that both Slctlp2 mRNA and protein were mainly present in the foregut and midgut of the 5th and 6th instar larvae during the feeding stages. In situ hybridization and immunohistochemistry confirmed that both Slctlp2 mRNA and protein were predominately present in the midgut. Expression of the gene was not induced by bacterial infection. Juvenile hormone III induced the gene expression, while 20-hydroxyecdysone had no impact on the expression. The expression of Slctlp2 mRNA and protein was down-regulated by starvation but up-regulated by re-feeding. The SlCTLP2 protein was detected in the lumen residues of the anterior, middle and posterior midgut and feces of the feeding 6th instar larvae, suggesting that it was secreted from the epithelium into the lumen of the gut. The results suggest that this Slctlp2 gene may be involved in digestive process of food proteins during the feeding stages of the larval development.

Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis

BackgroundWing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.ResultsIn total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated.ConclusionsExtensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

De novo characterization of transcriptome and gene expression dynamics in epidermis during the larval-pupal metamorphosis of common cutworm.

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, β-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and β-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.