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Gynaephora qinghaiensis (Lepidoptera: Lymantriidae: Gynaephora), a serious economic pest in alpine meadows, is mainly distributed in Yushu prefecture, Qinghai province, China. In this study, we aimed to investigate the genetic diversity and population structure of G. qinghaiensis through analyzing the sequence of 194 mitochondrial cytochrome oxidase subunit (COI) genes (658 bp in length) identified from 10 geographic populations located in three different countries, including Zhiduo, Zaduo, and Chengduo, of Yushu prefecture. Eleven haplotypes were identified from all populations of G. qinghaiensis with high levels of haplotype diversity (0.78500) and low levels of nucleotide diversity (0.00511). High levels of genetic differentiation and low levels of gene flow were also detected among the populations of G. qinghaiensis. Analysis of molecular variance (AMOVA) showed that 90.13% of the variation was attributed to distribution among groups (Chengduo, Zhiduo, and Zaduo), and 5.22% and 4.65% were, respectively, attributed to distribution among populations, within group, and within populations. The result of mantel test showed a highly significant positive correlation (P

Gynaephora qinghaiensis is a pest on the Qinghai-Tibet Plateau (QTP) that has led to substantial destruction of grassland vegetation. Its pupae are also natural hosts of parasitic wasp. Sexual dimorphism in immune responses is prevalent in vertebrates and invertebrates. However, sexual dimorphism in immune responses of insects, particularly G. qinghaiensis, is poorly understood at the transcriptional level. Here, we performed transcriptome sequencing in male and female pupae of G. qinghaiensis. A total of 118,357,040 clean reads were obtained and assembled into 114,944 unigenes. To explore the difference of immune responses in pupae of both sexes (male and female) of G. qinghaiensis. based on transcriptional level, we characterized the expression profiles of candidate transcripts in the two sexes of G. qinghaiensis. A total of 3,469 unigenes (1,888 up-regulated and 1,581 down-regulated genes) were differentially expressed in pupae of both sexes. Among the differentially expressed genes (DEGs), 263 unigenes related to immune responses were found in the two sexes in G. qinghaiensis; of these, 202 were up-regulated and 61 were down-regulated in the female pupae compared to male pupae. This indicates there were some differences in the expression of immune-related genes between male and female G. qinghaiensis pupae. Therefore, we speculated that the differences in immune responses also likely exsited in pupae of both sexes. Our report provides a valuable genomic resource for further studies of Gynaephora and improves our understanding of the molecular mechanisms underlying immunological differences between male and female insects.

Ophiocordyceps sinensis, a Chinese complementary and alternative medicine (CAM), is an entomopathogenic, fungus, parasitizing larvae of the moth genus Thitarodes. It has three stages of the life cycle, i.e., the anamorph mycelia prior to infection (Cm_Os), the mycelia sclerotium forming in the caterpillar (Te_Ca), and the fruiting bodies or stromata (Te_St). Characterization of the O. sinensis transcriptome among these stages could provide a better understanding of the underlying biology processes. Transcriptomics of the O. sinensis asexual mycelia and hyphae in deceased caterpillars and perithecial stroma was assessed by using Illumina HiSeq™ 2000 technology. A total of 14,922 unigenes were identified and categorized into 46 sub-categories under three gene ontology categories ("biological process", "cellular component", and "molecular function"). Of these genes, 5520 were differentially expressed among the libraries of these three groups of samples (P 0.05), and 391 genes occurred in all three groups. Compared to the anamorph stage, there were 3049 differentially expressed genes (DEGs) in the teleomorph stage, but only 1023 DEGs occurred within the teleomorph groups (Te_St vs. Te_Ca). Collectively, this study provides a novel resource to further investigate O. sinensis and their three different development stages.

As a pest on the Qinghai-Tibet Plateau, Gynaephora qinghaiensis causes severe damage to grassland vegetation and its pupae are also natural hosts of Thektogaster sp. To successfully parasitize, endoparasitoids generally introduce or secrete multiple parasitic factors into the host body during the spawning stage to suppress the host immune response. To study the parasitic effects of Thektogaster sp. on G. qinghaiensis, a transcriptome analysis of immune-related genes in parasitized and nonparasitized G. qinghaiensis pupae was performed. A total of 371,260,704 clean reads were assembled into 118,144 unigenes with an average length of 884.33 base pairs. Of these, 23,660 unigenes were annotated in at least one database and 94,484 unigenes were not annotated in any databases. These findings indicated that the majority of the genetic resources (79.97% of all unigenes) in Gynaephora should be further explored. Parasitization significantly affected the transcriptional profile of G. qinghaiensis pupae. The present study identified 12,322 differentially expressed genes and 57 immune-related genes were identified in parasitized G. qinghaiensis pupae. Most immune-related genes were downregulated, potentially resulting from the inhibitory effect of Thektogaster sp. on G. qinghaiensis pupae after parasitization. Overall, the transcriptome analysis sheds valuable light on the molecular mechanisms of G. qinghaiensis parasitization by Thektogaster sp. and promotes the development of novel biocontrol strategies for Gynaephora based on immune defense.

Ophiocordyceps sinensis, also referred to as the Chinese caterpillar fungus, is a rare entomopathogenic fungus found in the Qinghai-Tibetan Plateau that is used as a traditional Chinese medicine. O. sinensis parasitizes the larvae of the ghost moth Thitarodes. Characterization of the transcriptome of O. sinensis before and after host infection may provide novel insight into the process by which the fungus interacts with Thitarodes and may help researchers understand how to sustain this valuable resource. In this study, we performed RNA-sequencing (RNA-seq) using Illumina HiSeqTM 2000 technology to generate gene expression profiles of two developmental stages of O. sinensis. Thread-like hyphae before infection and yeast-like hyphal bodies after infection of host larvae were collected for transcriptome analysis. We found that 1640 genes were differentially expressed (q-value

Ophiocordyceps sinensis, an entomopathogenic fungus of the Tibetan Plateau is one of the most valuable traditional Chinese medicines. A fungal pathogen of O. sinensis, Polycephalomyces sp. GIMCC 3.570 (isolate 2010A-0818) was characterized by morphology, related to sequences of six genes (SSU, ITS, LSU, TEF1-α, RPB1 and ATP6) found in a BLAST search and five genes (SSU, LSU, TEF1, RPB1 and RPB2) by phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods. The results strongly support placement of P. sp. GIMCC 3.570 in the genus Polycephalomyces. To determine the influence of P. sp. GIMCC 3.570 on the reproduction of its host, 30 artificially infected O. sinensis samples were compared with 30 uninfected controls. In the infected fungus, the life span of the stroma was significantly shorter and significantly fewer ascospores were produced compared with uninfected O. sinensis (p

Thitarodes jiachaensis is a host species of the pathogenic caterpillar fungus Ophiocordyceps sinensis, which is a fungus with broad medical effects and great economic value. Curated genomic information on Thitarodes is still limited, and the interaction between host Thitarodes larvae and O. sinensis during infection is incompletely understood. In this study, we performed transcriptome sequencing for T. jiachaensis before and after O. sinensis infection using the Illumina sequencing platform, and we identified the transcripts associated with the defense response of T. jiachaensis upon O. sinensis infection. A total of 161,804 transcripts and 94,827 unigenes for T. jiachaensis were obtained from 26.62-Gb clean reads, and 35.03% of all the unigenes were annotated in current databases. The expression of 1581 genes was significantly altered following infection; among them, 928 (58.70%) were up-regulated and 653 (41.30%) were down-regulated. Genes encoding physical barriers such as cuticle proteins and peritrophic matrix proteins, antimicrobial peptides (AMPs), pattern recognition receptors (PRRs), and enzymes in the proteolytic cascade were predicted to be involved in the response of T. jiachaensis to O. sinensis infection. Together, these data provide a valuable genomic resource for further studies of Thitarodes and increase our understanding of the host-pathogen interaction that occurs between Thitarodes and O. sinensis.

The wolf spider Pardosa pseudoannulata is a dominant predator in paddy ecosystem and an important biological control agent of rice pests. Temperature represents a primary factor influencing its biology and behavior, although the underlying molecular mechanisms remain unknown. To understand the response of P. pseudoannulata to temperature stress, we performed comparative transcriptome analyses of spider adults exposed to 10°C and 40°C for 12 h. We obtained 67,725 assembled unigenes, 21,765 of which were annotated in P. pseudoannulata transcriptome libraries, and identified 905 and 834 genes significantly up‐ or down‐regulated by temperature stress. Functional categorization revealed the differential regulation of transcription, signal transduction, and metabolism processes. Calcium signaling pathway and metabolic pathway involving respiratory chain components played important roles in adapting to low temperature, whereas at high temperature, oxidative phosphorylation and amino acid metabolism were critical. Differentially expressed ribosomal protein genes contributed to temperature stress adaptation, and heat shock genes were significantly up‐regulated. This study represents the first report of transcriptome identification related to the Araneae species in response to temperature stress. These results will greatly facilitate our understanding of the physiological and biochemical mechanisms of spiders in response to temperature stress.

Ophiocordyceps sinensis (syn. Cordyceps sinensis), one of the most valuable medicinal mushrooms, has great economic importance on the Tibetan Plateau. We isolated an anamorphic fungus Lecanicillium pui from natural O. sinensis specimens and found that the optimal temperature for its culture on potato dextrose agar media was 25°C. Cell ultrastructure in L. pui hyphae and spores was characterized by transmission electron microscopy, and it was observed that some primary organelles showed the typical fungal features. Five chemical elements were determined in this fungus and niobium was discovered for the first time even with trace amounts. A species-specific method, nested polymerase chain reaction, was established to investigate the colonization of this fungus. Thus, the extensive distribution of L. pui on O. sinensis, in the shape of hyphae or mycelia, suggested that it may have subtle and chronic effects on the growth of the O. sinensis teleomorphic stage. These findings provide a potential reference, in the view of microbial ecology, for the study on the occurrence and mechanism of development of O. sinensis.

Thitarodes pui larvae are the hosts of a medicinal fungus, Ophiocordyceps sinensis, and are naturally distributed at an altitude of 4100-4650 m on Segrila Mountain of the Tibetan Plateau. Here, we conducted transcriptome profiling of T. pui larvae based on the Illumina high-throughput sequencing platform. Subsequently, we explored elevation-based differences of T. pui by comparing gene expression profiles between larvae at high-altitude (natural conditions) and larvae exposed to short-term (2months) low-altitude conditions. A total of 105,935,208 clean reads were assembled into 70,048 unigenes with a mean length of 639 bp. All unigenes were searched against public databases, with 51.26% unigenes being successfully annotated in the NR, SWISS-PROT, EuKaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genome (KEGG) databases. A total of 11,846 unigenes were functionally classified into 239 KEGG pathways. Metabolism was the most represented pathway, with 4271 unigenes (36.05%). Using the transcriptome data as a reference, 629 (311 up-regulated/318 down-regulated) genes were differentially expressed by low-altitude larvae when compared with those of high-altitude larvae. The most significantly differentially expressed genes were annotated in the processes of carbohydrate metabolism, lipid metabolism, and respiration. This report provides valuable information about the T. pui transcriptome for future genomic studies, including how gene expression is altered in larvae reared at different elevations.

Ophiocordyceps sinensis, a well-known Chinese complementary herb, is a rare and valuable therapeutic resource. Cordyceps militaris (C. militaris) is a commonly used substitute for O. sinensis. A metabolomic-based approach for exploring the similarities and differences in the metabolites of O. sinensis and C. militaris in water-boiled and 50% ethanol-soaked extracts is of great significance. To distinguish between the global metabolite profiles of O. sinensis and C. militaris extracts obtained from either the water-boiled or 50% ethanol-soaked methods, we investigated the herb samples using 1HNMR-based metabolic fingerprints combined with multivariate statistical analysis. This study revealed that a total of 52 primary metabolites were identified and quantified from O. sinensis and C. militaris samples. Forty-three (83% of 52) metabolites were detectable in both O. sinensis and C. militaris. According to the variable importance in projection (VIP) value and p-value from the Mann-Whitney test, 7 metabolites (alanine, aspartate, glutamate, mannitol, ornithine, serine, and trehalose) differed between O. sinensis and C. militaris. Arginine, glucose, putrescine, pyroglutamate, betaine, O-phosphocholine, and xylose differed significantly between the water-boiled and 50% ethanol-soaked methods used to prepare the herb extracts. This study demonstrated that water-boiled extraction was a much faster method (30 min. vs 360 days) that resulted in a 30% higher number of extracted metabolites (compared to 50% for the ethanol-soaked method) for both O. sinensis and C. militaris.

Ophicordyceps sinensis is a well-known traditional Chinese medicine and cultured mycelium is a substitute for wild O. sinensis. Metabolic profiles of wild O. sinensis from three geographical locations and cultivated mycelia derived from three origins were investigated using (1)H nuclear magnetic resonance (NMR) analysis combined with multivariate statistical analysis. A total of 56 primary metabolites were identified and quantified from O. sinensis samples. The principle component analysis (PCA) showed significant differences between natural O. sinensis and fermentation mycelia. Seven metabolites responsible for differentiation were screened out by orthogonal partial least squares discriminant analysis (OPLS-DA). The concentrations of mannitol, trehalose, arginine, trans-4-hydroxyproline, alanine and glucitol were significantly different between wild and cultured groups. The variation in metabolic profiling among artificial mycelia was greater than that among wild O. sinensis. Furthermore, wild samples from different origins were clearly distinguished by the levels of mannitol, trehalose and some amino acids. This study indicates that (1)H NMR-based metabolomics is useful for fingerprinting and discriminating O. sinensis of different geographical regions and cultivated mycelia of different strains. The present study provided an efficient approach for investigating chemical compositions and evaluating the quality of medicine and health food derived from O. sinensis.

In the present study, we extracted the chitosan from the larvae of blowfly Chrysomya megacephala, a new source of insect chitosan, using chemical methods. We evaluated the physical properties of the blowfly chitosan using a variety of approaches, including preliminary color-change identification, molecular weight determination, elemental analysis (EA), Fourier transform infrared spectroscopy (FTIR), solid-state (13)C cross-polarization and magic-angle-spinning nuclear magnetic resonance spectroscopy ((13)C CP/MAS NMR) and scanning electron microscopy (SEM). Its antioxidant property was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays. The results showed that the molecular weight of the blowfly chitosan (501 kDa) was lower than that of the commercial chitosan (989 kDa), and its degree of deacetylation (DDA) (87.9-89.6%) was also higher than that of the commercial chitosan (83.8-85.8%). Furthermore, the blowfly chitosan exhibited excellent antioxidant activity and its IC50 value was 1.2 mg/ml. Therefore, the blowfly larvae could be a novel alternative source of chitosan and might be used as a natural antioxidant.

Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10of the initial amount of O. sinensis genomic DNA (r2= 0.999) over 7 orders of magnitude (4 × 101to 4 × 107fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P < 0.05). Our protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

KEY MESSAGE : Silencing OsMPK3 decreased elicited JA levels, which subsequently reduced levels of herbivore-induced trypsin protease inhibitors (TrypPIs) and improved the performance of SSB larvae, but did not influence BPH. Mitogen-activated protein kinases (MPKs) are known to play an important role in plant defense by transferring biotic and abiotic signals into programmed cellular responses. However, their functions in the herbivore-induced defense response in rice remain largely unknown. Here, we identified a MPK3 gene from rice, OsMPK3, and found that its expression levels were up-regulated in response to infestation by the larvae of the striped stem borer (SSB) (Chilo suppressalis), to mechanical wounding and to treatment with jasmonic acid (JA), but not to infestation by the brown planthopper (BPH) Nilaparvata lugens or to treatment with salicylic acid. Moreover, mechanical wounding and SSB infestation induced the expression of OsMPK3 strongly and quickly, whereas JA treatment induced the gene more weakly and slowly. Silencing OsMPK3 (ir-mpk3) reduced the expression of the gene by 50-70 %, decreased elicited levels of JA and diminished the expression of a lipoxygenase gene OsHI-LOX and an allene oxide synthase gene OsAOS1. The reduced JA signaling in ir-mpk3 plants decreased the levels of herbivore-induced trypsin protease inhibitors (TrypPIs) and improved the performance of SSB larvae, but did not influence BPH. Our findings suggest that the gene OsMPK3 responds early in herbivore-induced defense and can be regulated by rice plants to activate a specific and appropriate defense response to different herbivores.

Thitarodes pui is one of the host species of the Chinese caterpillar fungus Ophiocordyceps sinensis as a traditional Chinese medicine with economic and medical importance. The pupal and adult stages of T. pui are sexually dimorphic. In order to elucidate the molecular mechanisms involved in the sexually dimorphic development of T. pui, we compared the transcriptomes of female and male pupae and adults. We obtained 15,881,734, 16,962,086, 17,514,743, and 17,770,904 clean reads from female pupae, male pupae, female adults, and male adults, respectively. The reads obtained from the four samples were pooled and assembled into 65,165 unigenes, 23,597 of which were annotated. Candidate genes involved in sexual development were identified and analysed. Gene expression analysis revealed that 1406 genes were differentially expressed in male and female pupae, 448 of which were up-regulated in males and 958 were up-regulated in females. A total of 2025 genes were differentially expressed in male and females adults, 1304 of which were up-regulated in males and 721 were up-regulated in females. The functional enrichment of the differentially expressed genes indicated that reproduction and cuticle synthesis were regulated differently between the sexes. The transcriptome data obtained provide significant information regarding the genes involved in sexually dimorphic development, which will improve our understanding of the molecular mechanisms related to sexual dimorphism and helpful for the moth mass rearing which would provide enough host insects for the sustainable utilization of O. sinensis.

By exposing the common cutworm Spodoptera litura Fabricius larvae to a range of Zinc (Zn) stress, we investigated the effects of dietary Zn on Zn accumulation, metallothionein (MT), and on the ultrastructure of the midgut. The techniques we used were inductively coupled plasma-atomic emission spectrometer (ICP-AES), real-time PCR combined with cadmium-hemoglobin total saturation, and transmission electron microscopy (TEM), respectively. There was a significant dose-response relationship between the Zn accumulations in the midgut of the larvae and the Zn concentrations in the diet. Furthermore, both MT content and MT gene expression in the midgut were significantly induced in the 50-500 mg Zn/kg treatments, and were significantly positively correlated with the Zn accumulations in the midgut. When S. litura larvae were fed with the diet treated with 500 mg Zn/kg, Zn accumulation and MT content in the midgut was 4450.85 mg Zn/kg and 372.77 mg/kg, respectively, thereafter there was a little increase; the level of MT gene expression was maximal, thereafter there was a sharp decrease. TEM showed that numerous electron-dense granules (EDGs) and vacuoles appeared in the cytoplasm of the midgut cells, their number and size being closely correlated with the Zn accumulations in the midgut. Moreover, the nuclei were strongly influenced by Zn stress, evidenced by chromatin condensation and irregular nuclear membranes. Therefore, after being exposed to Zn in the threshold (500 mg Zn/kg) range, S. litura larvae could accumulate Zn in the midgut, which led to the induction of MT and changes in cell ultrastructure (mainly the presence of EDGs). The induction of MT and precipitation of Zn in EDGs may be the effective detoxification mechanisms by which the herbivorous insect S. litura defends itself against heavy metals.

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ∼15 × 8 μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2–17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17 ± 0.73 × 6.49 ± 0.4 μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.

In insects, P450s are responsible for the oxidative metabolism of structurally diverse endogenous and exogenous compounds including plant allelochemicals and insecticides. A novel full-length P450 cDNA, CYP6B47, was cloned from Spodoptera litura (Lepidoptera: Noctuidae). The sequence is 1718 bp in length with an ORF of 1509 bp encoding 503 amino acid residues. The phylogenetic analysis indicated that CYP6B47 belongs to CYP3 clan and second clade of CYP6Bs which contain 11 P450s from Noctuidae. Quantitative real-time PCR showed that CYP6B47 was expressed only in larvae stages and had a high level of transcription in the midgut and fat body. In addition, we cloned a 2141-bp 5'-flanking regions and presented the basal luciferase activities of promoter. We also predicted multiple putative elements for transcription factors binding in the 5'-flanking region. Interestingly, the expression of CYP6B47 significantly increased in the midgut and fat body after lead (Pb) exposure for 5 generations. Larvae tolerance to the alpha-cypermethrin (35% increased in LC(50)) and fenvalerate (52% increased in LC(50)) were improved after pre-exposure to 50 mg/kg Pb. These dates suggested that lead increased tolerance of larvae to insecticides mainly through transcriptional induction of detoxification genes including CYP6B47.

Beta-1,3-glucan recognition proteins (βGRPs), as a class of pattern recognition receptors, are involved in activation of the immune response in invertebrates. We cloned two cDNAs encoding putative βGRPs from larvae of Thitarodes pui, a host species of Ophiocordyceps sinensis with great economic importance in the Tibetan Plateau. The two putative βGRPs were phylogenetically classified into a novel clade 4, and designated TpβGRP-4a and TpβGRP-4b, respectively, with calculated molecular masses of 53,265 and 43,991 Da. Both TpβGRPs contained a C-terminal domain with sequence similarity to β-1,3-glucanases but without the glucanase active site. TpβGRP-4b markedly differed from other family members including TpβGRP-4a in the N-terminal region by a large deletion of ∼80 amino acid residues. Homology modelings revealed an eight-stranded β-sandwich fold (β1-β8) and two β-strands (only β1 and β2), respectively, in the N-terminal domains of TpβGRP-4a and -4b. TpβGRPs showed similar developmental expression patterns in fat body. TpβGRP-4a and -4b transcripts were induced highest 313- and 16-fold, respectively, in resistant 8th instar larvae challenged with conidia of entomopathogenic fungus Beauveria bassiana. By contrast, significant reductions in TpβGRPs expression were observed in conidia-injected susceptible 6th instar larvae (compared with saline-injected controls), accompanied by production of hyphal bodies in hemolymph. These results suggest that TpβGRPs might contribute to host defense against fungal infection, and TpβGRP-4b with the unusual deletion of the N-terminal region might have evolved new functions for βGRP family proteins.

Apolipophorin III (apoLp-III) functions in lipid transport and immune activation in insects. We cloned a cDNA encoding putative apoLp-III from larvae of Thitarodes pui, a host species of Ophiocordyceps sinensis, with great economic importance in the Tibetan Plateau. Excluding a putative signal peptide of the first 20 amino acid residues, the 171-residue mature apoLp-III has a calculated molecular mass of 18,606 Da. T. pui apoLp-III shares little sequence homologies (

By exposing Spodoptera litura Fabricius larvae to nickel (Ni) in artificial diets for successive three generations, we investigated the impacts of the dietary Ni on growth and immune response of the fifth and sixth instar larvae at 24 h intervals. The time of newly moulted fifth instar larvae was labelled as 0 h. After exposure to 5 mg/kg Ni for two generations, Ni exposure significantly improved larval phenoloxidase activity and encapsulation grade in fifth instar larvae when compared to controls, except for encapsulation grade at 72–120 h in the second generation. However, higher concentrations of Ni (≥10 mg/kg) only significantly reduced encapsulation grade at 72–120 h. In the third generation, insects given higher dietary levels of Ni (≥10 mg/kg) showed lower immune responses and retarded relative growth rate (RGR) compared to controls, but those exposed to lower Ni levels (≤5 mg/kg) had a significantly improved encapsulation grade at 24–72 h. Larvae at lower Ni level (≤5 mg/kg) treatments had significantly higher RGR in comparison with that in controls. There was no significant difference in food relative consumption rate (RCR) and RGR among any treatment of the fifth instar larvae in three successive generations. These results indicated that the type and extent of effects on growth and immune responses of S. litura varied with the Ni concentrations and exposure periods.

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) superfamily, and is an important carrier for the uptake of vitellogenin (Vg) into developing oocytes of all oviparous species. The first full-length message for a VgR from a Lepidopteran insect was cloned and sequenced from the ovary of Spodoptera litura Fabricius (GenBank accession no. GU983858). The coding region consisted of 5370 bp flanked by a 49 bp 5'-untranslated region (UTR) and a 177 bp 3'-UTR, which encoded a 1798-residue protein with a predicted molecular weight (MW) of 201.69 kDa. S. litura VgR (SlVgR)comprised two ligand binding sites with four LDLR class A repeats in the first domain and seven in the second domain, an epidermal growth factor-like domain containing an LDLR class B repeat and a YWXD motif, a transmembrane domain and a cytoplasmic domain. A phylogenetic relationship placed SlVgR as a separate group from the other insects. SlVgR messenger RNA (mRNA) was specifically expressed in the ovarian tissues. The developmental expression patterns showed that VgR mRNA was first transcribed in 6(th) day female pupae and the maximum level of VgR mRNA appeared in 36-h-old adults. Immunoblot analysis detected an ovary-specific VgR protein with a MW of ∼200 kDa, whose development profiles were consistent with VgR mRNA expression patterns. RNA inteference (RNAi) specifically disrupted the VgR gene by injection of 3 or 5 µg VgR double-stranded RNA per insect in 4(th) or 6(th) day pupae. RNAi of SlVgR led to a phenotype characterized by high Vg accumulation in the haemolymph, low Vg deposition in the ovary and the failure of insect spawning. These results mean that VgR is critical for binding Vg and transporting it into the oocytes of the insect ovary, thus playing an important role in insect reproduction.

Effects of dietary nickel on apoptosis of hemocytes of the 6th instar larvae of Spodoptera litura Fabricius were evaluated using flow cytometry and Laser Scanning Confocal Microscopy (LSCM) by feeding S. litura larvae with artificial diets amended with different concentrations of nickel (1, 5, 10, 20 and 40 mg/kg) for 3 generations. Results showed that the hemocyte apoptosis rate of the 1st generation larvae increased with the increasing nickel concentrations in diet, and the apoptosis rate in the treatment with 40 mg/kg nickel was significantly higher than that in other treatments. In the 2nd generation, the highest hemocytes apoptosis rate was recorded in the treatment of the larvae exposed to 5 mg/kg nickel. The apoptosis rates in the larvae stressed by higher concentrations of nickel were lower than that in control. The apoptosis rates in the larvae exposed to nickel for 3 generations showed similar concentration-responses with that in the 2nd generation. However, the apoptosis rates in the larvae stressed with higher levels of nickel were all significantly inhibited in comparison with that in control. Therefore, effects of nickel stress on apoptosis of S. litura larvae were related with the nickel concentration in diets and the larval generations that were stressed by nickel.

Nickel accumulated in midugt of Spodoptera litura Fabricius could induce the expression of metallothionein, one of the most important detoxification proteins in organisms. In the present study, the effects of dietary nickel on the activities of detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST) in the midgut of S. litura larvae have been studied to get an understanding of the detoxification mechanisms of S. litura larvae to excessive nickel. Results showed that CarE activities in the midgut of the 5th instar larvae decreased at lower levels of nickel (⩽5 mg/kg), while increased with increasing nickel doses at higher levels of nickel (⩾10 mg/kg) exposure in successive 3 generations. CarE activities of the 6th instar larvae were also characterized as inhibited at low levels of nickel exposure, and improved at higher levels in the 1st generation. CarE activities of 6th instar larvae in the 2nd and 3rd generations were all lower than that in control. However, GST activities in the midgut of the 5th and 6th instar larvae all increased with increasing nickel doses (1–20 mg/kg) in diets.

In order to understand the composition and quantitative variation of ecdysteroids in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest, we analyzed the ecdysteroid composition and titers in the adult and egg of this pest. On day 4 after eclosion, the titer of ecdysteroids in the male adult was 0.080 ng/adult, much lower than 5.978 ng/adult in the female adult. During the development of ovaries, the titer of ecdysteroids was low on the first two days, and high in the late period, with the peak (10.48 ng/ovary) appearing on day 3. During the development of eggs, the titer of ecdysteroids was about 0.010 ng/egg from day 1 to day 3, and then decreased to 0.006 ng/egg on day 4. In both adults and eggs, three main components of ecdysteroids were found by identification of HPLC/RIA. They were 20-hydroxyecdysone, 26-hydroxyecdysone, and an unidentified component.

Metallothionein expression induced by nickel accumulation in the midgut of Spodoptera litura Fabricius larvae was investigated by exposing S. litura larvae to the artificial diets amended with different doses of nickel for 3 generations. Nickel accumulations in the midgut of 6th instar larvae were detected by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), and the induced metallothionein expression in the midgut of 5th and 6th instar larvae in 120 h were quantified using cadmium-hemoglobin total saturation methods. The accumulated nickel in the midgut of the 6th instar larvae within a generation increased with the increase of the nickel doses in the treated diets and showed significant dose-dependence with the nickel doses in diets. Metallothioneins induced by nickel accumulated in the larval midgut also increased with nickel doses in diets and with the increasing stressing time in a generation. Results also suggested that the metallothionein expression was different with the time of S. litura larvae stressed by nickel.

The artificial diets mixed with various concentrations of nickel were offered to the larvae of the phytophagous insect Spodoptera litura Fabricius for 3 generations. Nickel accumulations in the 6th instar larvae, pupae and newly emerged adults of the corresponding generations of S. litura were investigated by inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), and the effects of nickel accumulations on the survival rate of S. litura were also evaluated by individual rearing. The results showed that nickel accumulated in the 6th instar larvae, pupae and adults of S. litura, and the accumulated nickel in all the tested developmental stages within a generation increased with the increase of the nickel doses in the treated diets and showed significant dose-dependent relationship with the nickel doses in the artificial diets. The results also indicated that the nickel accumulations in the 6th instar larvae, pupae, and newly emerged adults from the 3rd generation were higher than those from the 2nd generation, which were also higher than those from the 1st generation. Nickel concentrations in pupae and adults were significantly lower than those in larvae, which indicated that the excessive nickel might be excreted during metamorphosis. Furthermore, larval survival rate, pupation rate and eclosion rate of S. litura in the tested three generations all decreased with the increase of the nickel doses in the treated diets.

In order to understand the composition and quantitative variation of ecdysteriods in the larvae of Opogona sacchari (Bojer), a invasive alien pest, we analyzed the larval ecdysteroid composition and titers in this pest. The main component of ecdysteriods in the larvae of O. sacchari is 20-hydroxyecdysone, and also there is a little 26-hhdroxyecdysone. The titer of ecdysteriods in the larvae from the 1st instar to the 7th instar was gotten higher gradually compared on ng ecdysteroid/larva, but no regularity could be found about the titer if compared on ng ecdysteroid/g avoirdupois. There was only one peak of ecdysteroids (0.5475 ng/larva) showed at day 2 during the developmental time of the 6th instar larvae. However, there were two peaks appearing during the developmental time of the 7th instar larvae, one peak (0.29415 ng/larva) at day 3, another (0.214 ng/larva) at day 5.

Thitarodes larvae are the host of Ophiocordyceps sinensis and exist in the permafrost region of the Tibetan Plateau.To understand the adaptation mechanism of Thitarodes larvae to seasonal fluctuations of ambient temperatures in the Tibetan Plateau by studying seasonal changes of the fatty acids composition in the larvae of T. pui.The profile of fatty acids in the 6th instar T. pui larvae collected at the mid-month in a whole year were examined by GC-MS.There was a negative correlation between the total lipid and ambient (soil) temperature. Further study indicated that oleic, palmitic, linoliec, palmitoleic, stearic were the major fatty acids. The ratio of unsaturated fatty acids to saturated fatty acids (U/S) and the unsaturated index (UI) in triacylglycerols remain stable during the whole year, while the U/S and UI in phospholipids vary dramatically in response to soil temperature.The fluctuations in phospholipids were attributed to seasonal changes of oleic and linoleic. The changes of the fatty acid composition may result from their adaptation to the variation of temperature in different seasons.

Ophiocordyceps sinensis is a well-known entomogenous and medicinal fungus. After its anamorphs parasitize the larvae of the genus Thitarodes, fruit-bodies may form to be used as medicine. However, its developmental mechanisms remain unknown. The distribution of O. sinensis was determined in different tissues of the Thitarodes larvae and the dominant plant species using real-time quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) technique, respectively. We found that more fungal material was located in plants than in larvae, especially in Ranunculus tanguticus. A considerable amount was detected in larval intestinal-wall and plant roots. It is suggested that plants are the potential hosts of O. sinensis, which modifies our understanding of the life cycle of O. sinensis and indicates that the phytophagous larvae may become infected as they feed. Our research may contribute to the study of systematic evolution and population ecology of O. sinensis, elucidate its developmental mechanism and promote sustainable harvesting.

Nickel is an environmental pollutant that adversely affects the male reproductive system. In the present study, the effects of nickel exposure on Spodoptera litura Fabricius were investigated by feeding larvae artificial diets containing different doses of nickel for three generations. Damage to testes and effects on male reproduction were examined. The amount of nickel that accumulated in the testes of newly emerged males increased as the nickel dose in the diet increased during a single generation. Nickel exposure increased the amount of thiobarbituric acid reactive substances and decreased the amount of glutathione in treatment groups compared with the control. The activity levels of the antioxidant response indices superoxide dismutases, catalase, and glutathione peroxidase in the testes showed variable dose-dependent relationships with nickel doses and duration of exposure. Nickel doses also disrupted the development of the testes by decreasing the weight and volume of testes and the number of eupyrene and apyrene sperm bundles in treatment groups compared with the control. When the nickel-treated males mated with normal females, fecundity was inhibited by the higher nickel doses in all three generations, but fecundity significantly increased during the second generation, which received 5 mg kg(-1) nickel. Hatching rates in all treatments significantly decreased in a dose-dependent manner in the three successive generations. The effects of nickel on these parameters correlated with the duration of nickel exposure. Results indicate assays of testes may be a novel and efficient means of evaluating the effects of heavy metals on phytophagous insects in an agricultural environment.

In order to understand the composition and quantitative variation of ecdysteriods in the pupae of Opogona sacchari (Bojer), a new invasive alien pest, we analyzed the pupal ecdysteroid composition and titers in this pest. The main component of ecdysteriods in the pupae of O. sacchari is 20-hydroxyecdysone. The titre of ecdysteriods in both male pupae and female pupae get higher gradually, and then decreased slowly after reaching a peak (80 ng/pupa). The titre of ecdysteriods was about 30 ng/pupa in the later stage of pupae. The peak of male pupae appeared on the third day, but the peak of female pupae lasted from the second day to the fourth day during pupal developments. The ecdysteroid content was higher in female pupae than in male pupae during specific pupal time (the 1st, 2nd, 4th, 5th, 6th day). We also found that the titre of ecdysteroids in posterior part of pupae was higher than in anterior part of pupae obviously.

In order to explore the role of heat shock proteins (Hsps) during thermal stress and development in the predatory mite Neoseiulus cucumeris (Oudemans), we cloned and characterized five full-length Hsp genes. We investigated the expression levels of these genes by quantitative real-time PCR. The five genes characterized here were NcHsp90, NcHsp75, NcHsp70, NcHsp60, and NcHsp40. These Hsps showed high sequence conservation and had greatest identity with heat shock proteins of Metaseiulus occidentalis and other mite and insect species. All five NcHsp genes showed changes in their levels of expression during development. Higher levels of expression were observed in adult females than in adult males, but there were no significant changes between pre-oviposition and post-oviposition stages in the females. NcHsp90, NcHsp75, and NcHsp70 expression levels were up-regulated after a heat shock, and the increases in NcHsp75 and NcHsp70 expression levels were maintained for at least 3 h. Up-regulation of NcHsp60 and NcHsp40 was not detected after 1 h at a high temperature (35-45 °C); however, a significant down-regulation was observed after 3 h heat exposure at 35 °C and 3 h recovery at 25 °C. Cold shock treatment (-5 to 15 °C) for 1 h did not acute elicit changes in the expression levels of any of the genes. At 5 °C, the expression levels of NcHsp90 significantly increased after 6 or 24 h exposure compared to the levels after 1 h exposure. Thus, expression of Hsp genes in N. cucumeris reflected developmental changes, sexual difference, and variable induced response to thermal stress. Increased expression of Hsps might protect N. cucumeris individuals under extreme temperature conditions. Therefore, it may be possible to enhance the thermal tolerance of commercially available N. cucumeris using temperature acclimation. Treatment at 35 °C should be suitable for such acclimation.

By adding different amount of zinc into the artificial medium of the insect larvae, the zinc-induced apoptosis of the larvae haemocytes of the herbivorous insectSpodoptera litura Fabricius was investigated with flow cytometer. The results showed that the increase of zinc dose in the artificial feed led to the accumulations of zinc in the larval hemolymph and fat body, and more zinc was accumulated in fat body than in hemolymph. The apoptosis of hemocytes was significantly induced at high zinc concentration (1000 mg·kg−1) in the insect diet, and the apoptosis rate was 63.63%, which was remarkably higher than that at control and lower concentrations (50-500 mg·kg−1). This suggests that the high dose of zinc in the artificial diet ofS. litura larvae could induce the apoptosis of the larval hemocytes ofS. litura.

The acceptance of 40 different strains of 24 Trichogramma (Hymenoptera: Trichogrammatidae) species for oak-silkworm host eggs, Antheraea pernyi Guerin-Meneville (Lepidoptera: Anthelidae) was tested in laboratory experiments. The oak-silkworm, which is commercially used in China to mass produce Trichogramma dendrolimi Matsumura on a large scale, was accepted for egg laying by 10 out of the 24 species tested but only 3 species (four strains of T. dendrolimi, three strains of T. chilonis Ishii and one strain of T. cacoeciae Marchal) successfully completed development to adult emergence. The number of adults emerged per host egg averaged 83.2, 37.0, 42.3, 53.0 for four different strains of T. dendrolimi; 42.5, 10.0 for two strains of T. chilonis; 24.5 and 0 for two strains of T. cacoeciae. Seven other Trichogramma species develoedp in A. pernyi eggs, but no adult emergence occurred and no emergence holes on the chorion were found. The number of Trichogramma larvae, pupae, and adults together per host egg aver...

The value of corn pollen as a food source for Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) was assessed in the laboratory by testing the effect of different pollen diets on the longevity, capacity of parasitism and other life-table parameters. T. brassicae females that fed on corn pollen and water (wet filter paper dusted with pollen) lived significantly longer (4.97 days) than females that fed on water alone (2.67 days), but significantly shorter than those feeding on honey alone (8.37 days) or corn pollen and honey (8.23 days), respectively, in the absence of host eggs. The presence of European corn borer eggs (Ostrinia nubilalis Huebner, Lepidoptera: Pyralidae) significantly increased longevity. With host eggs available, female T. brassicae that fed on corn pollen and water lived significantly longer (4.9 days) than on water alone (2.60 days), but significantly shorter than on honey alone (12.33 days) or corn pollen and honey (12.17 days), respectively. The cumulative lifetime fecun...

The expression patterns of eight defenserelated genes in the herbivore-infested and jasmonatetreated (jasmonic acid, JA and its derivative MeJA) rice leaves were analyzed using RT-PCR. The results showed thatSpodoptera litura Fabricius (Lepidoptera: Noctuidae) herbivory induced the expression of lipoxygenase (LOX) and allene oxide synthase (AOS) genes that are involved in the jasmonate-signaling pathway. Moreover,S. litura damage resulted in the expression of farnesyl pyrophosphate synthase (FPS), Bowman-birk proteinase inhibitor (BBPI), phenylalanine ammonia-lyase (PAL) and other rice defenserelated genes that were also induced by aqueous JA treatment or gaseous MeJA treatment. These indicated that in rice leaves, the JA-related signaling pathway was involved in theS. litura-induced chemical defense. Mechanical damage and brown planthopper (BPH),Nilaparvata lugens (Stal) (Homoptera: Delphacidae) damage induced the expression ofLOX gene, but both treatments did not induce the expression ofAOS gene. However, BPH damage induced the expression of acidic pathogen-related protein 1 (PR1a), Chitinase (PR-3), andPAL genes, which is involved in the salicylatesignaling pathway. It was suggested that salicylate-related signaling pathway or other pathways, rather than jasmonate-signaling pathway was involved in the BPH-induced rice plant defense.

An enzyme-linked immunosorbent assay (ELISA) has been developed to quantify predation by Ummeliata insecticeps Boes. et Str. on rice planthoppers in paddy fields in Dasha Township, Guangdong Province. The assay was completely specific for rice planthopper materials. The detection periods for antigens after an adult of U. insecticeps had fed on three 3–5th instar nymphs of white-back planthopper (WBPH), Sogatella furcifera (Horvath), and brown planthopper (BPH), Nilaparvata lugens (Stal), at room temperature were 96 h and 120 h, respectively. The proportion of individual species of predators scoring positive ranged from 19. 05% to 47. 34% for WBPH, and from 9. 25% to 66. 67% for BPH in the early rice season. Although the numbers of the rice planthoppers consumed by U. insecticeps increased with increasing prey densities, the predation rates declined. When rice planthopper densities were low, the predation rates were relatively high. This kind of predation can explain why the rice planthoppers have never reached outbreak levels in rice fields in Dasha Township since 1973.

Ether extracts from a resistant rice variety, Jingxian 89, and a susceptible variety, Qidaizhan, were significantly toxic to Nilaparvata lugens adult females, causing 98% and 73.5% mortalities, respectively. The area covered honeydew droplets excreted by N. lugens adult females while feeding on Qidaizhan was 28.5-fold more than the area of those feeding on the resistant variety, Jingxian 89. The honeydew covered areas were reduced 29.7- and 8.8-fold, respectively, after feeding on plants treated with the ether extracts from both varieties. Among the fractions from each variety, fractions 9 (three sterols) from both varieties and fraction 10 (3-nitraphthalic acid) from Jingxian 89 showed significant repellent effects on the planthoppers. Further bioassays revealed that fraction 9 from extracts of both varieties at 0.02 mg/ml or higher deterred the feeding activity of the planthoppers, whereas the deterrent effects of fraction 10 from the extract of Jingxian 89 were 100- to 500-fold stronger than those of fraction 9. Fraction 10, identified as 3-nitraphthalic acid, unique in Jingxian 89, was extremely toxic to N. lugens adult females, and the LC50 and LC90 were 0.00045 and 0.00525 mg/ml, respectively. After extensive bioassays and analysis, we concluded that 3-nitraphthalic acid plays a key role in the resistance to N. lugens adult females.

Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae) is one of the most widely used and important biological control agents for thrips and other small pests worldwide. In the present study, we cloned two cDNAs of vitellogenins (Vgs, NcVg1 and NcVg2) and analyzed the effect of food source on the expression of both Vgs and fecundity in female adults. NcVgs showed higher sequence similarity to Vgs from Parasitiformes. Both neighbor-joining and maximum likelihood methods for phylogenetic analysis of NcVgs yielded similar topologies and showed that the Parasitiformes except Haemaphysalis longicornis segregated into a single clade that was separated into two subclades including one of both Vgs from N. cucumeris. Both transcripts, NcVg1 and NcVg2 revealed similar trends during developmental periods and reached the maximum level at the pre-oviposition period. When fed with different food sources, both NcVg1 and NcVg2 of female adults demonstrated a significant difference (P < 0.05) during the pre-oviposition period. Meanwhile, a positive correlation between the expression of Vgs and fecundity was observed. Therefore, the nutrients provided by the food sources affected fecundity resulting in differential expression of Vgs. Vitellogenin expression can be used as a molecular marker of fecundity of N. cucumeris.

Ophiocordyceps sinensis , one of the most important income sources of rural Tibetan families, is an entomopathogenic fungus that parasitizes the ghost moth Thitarodes larvae, which live in alpine meadows on the Tibetan Plateau and in the Himalayas. The annual yield of O. sinensis has gradually declined in recent years. However, there is no effective method to sustain or increase the yield of O. sinensis artificially because the life cycle of the O. sinensis anamorph remains unclear. Here we detected O. sinensis in alpine plant roots by nested-touchdown polymerase chain reaction (PCR). Forty-two alpine plant species were screened. The roots from 23 alpine plant species (54.76 %) tested positive including 13 families and 18 genera. The detection results indicate that O. sinensis is present in the plant roots during the anamorph life cycle, to deal with harsh conditions in alpine habitats and have an increased opportunity to infect the larvae. The finding provides new information regarding the biology and ecology of O. sinensis that may be used to sustain this valuable resource.

Background Chrysomya megacephala (Fabricius), a prevalent necrophagous blowfly that is easily mass reared, is noted for being a mechanical vector of pathogenic microorganisms, a pollinator of numerous crops, and a resource insect in forensic investigation in the postmortem interval. In the present study, in order to comprehensively understand the physiological and biochemical functions of C. megacephala, we performed RNA-sequencing and digital gene expression (DGE) profiling using Solexa/Illumina sequencing technology. Methodology/Principal Findings A total of 39,098,662 clean reads were assembled into 27,588 unigenes with a mean length of 768 nt. All unigenes were searched against the Nt database, Nr database, Swiss-Prot, Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genome (KEGG) with the BLASTn or BLASTx algorithm (E-value

Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the C T values (R (2)=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came body-wall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these results show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.