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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.

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

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.

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.

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.

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 (

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.

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.

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.

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...

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.

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.

Vitellogenin (Vg) cDNA from Spodoptera litura Fabricius was cloned and sequenced. The open reading frame (ORF) of Vg cDNA was 5247 nucleotides in length (GenBank Accession no. EU095334), which encoded for a protein of 1748 amino acids. S. litura Vg comprised three conserved regions (Vitellogenin-N domain, DUF1943 and von Willebrand factor type D domain (VWD)), a 17 amino-acid signal peptide and a RXXR cleavage signal (RTIR). The highly conserved GL/ICG motif, the DGXR motif and cysteine residues were found in the C-terminus of the Vg. Vg mRNA was found specifically in the female fat body. Vg expression was first transcribed in 6th day female pupae and levels increased with insect development. The maximum level of Vg mRNA appeared in 24-h-old adults. When S. litura larvae were exposed to lead (Pb) (25-200 mg Pb/kg), there was a significant inhibition in Vg of female adults. The start of Vg expression was advanced ahead by Pb, from 6th day pupae to 3rd day or 4th day pupae. Low levels of Vg in male adults were also induced by low concentrations of Pb (12.5 and 25 mg Pb/kg). These data show that Pb stress elicits an important Vg response in S. litura.

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300-750mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.

It has been suggested that herbivore would react to volatiles produced by herbivore infested plant due to potential change, either positive or negative, in the acceptability of the host plant. This hypothesis was tested for the brown planthopper (BPH) in the laboratory. Sixteen components of the headspace volatiles from rice seedlings with different treatments were collected with SPME and Tenax-TA trap and analyzed with GC and GC-MS. Significant differences in volatile emissions were observed for rice plants with different treatments. Undamaged control plants, mechanically damaged plants and the plants infested by BPH for 1 or 2 d emitted much lower amounts of volatiles compared to the plants infested by BPH for 3 or 5 d. The plants infested by BPH for 3 or 5 d emitted several volatiles that were not detected in undamaged control plants, mechanically damaged plants or the plants infested by BPH for 1 or 2 d. Spodoptera litura infested plants released much higher amounts of volatiles than those in all other treatments, and the contents of several green leaf volatiles, methyl salicylate and terpenoids increased dramatically. In dual-choice flight tunnel experiments, adult BPH females showed no significant preference between the untreated healthy plants and mechanically damaged plants or the plants infested by BPH adult females. However, rice plants damaged by S. litura had a clearly repellent effects on BPH adult females compared to healthy undamaged plants, mechanically damaged plants or the plants infested by BPH.