Profile of Ophiocordyceps sinensis transcriptome and differentially expressed genes in three different mycelia, sclerotium and fruiting body developmental stages.

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.

Cytological Characterization of Anamorphic Fungus Lecanicillium pui and Its Relationship with Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

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.

Transcriptome analysis of Ophiocordyceps sinensis before and after infection of Thitarodes larvae.

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 < 0.05), of which 818 were upregulated (49.878 %) and 822 were downregulated (50.122 %). Gene ontology (GO) analysis revealed that the differentially expressed genes (DEGs) were especially enriched in terms associated with Biological Process and Molecular Function. Several genes encoding transporter and permease proteins, three glycoside hydrolases, two mycotoxin-related proteins, an antigen protein, and an allergen were identified as being significantly up- or downregulated. Collectively, our findings provide a novel resource for understanding O. sinensis during two critical developmental stages, and offer the opportunity to further investigate the functional mechanisms underlying these stage-specific molecular differences.

Transcriptome characterization and gene expression analysis related to sexual dimorphism in the ghost moth, Thitarodes pui, a host of Ophiocordyceps sinensis.

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.

Metabolic characterization of natural and cultured Ophicordyceps sinensis from different origins by 1H NMR spectroscopy.

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.

Analysis of the transcriptome of blowfly Chrysomya megacephala (Fabricius) larvae in responses to different edible oils.

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<0.00001) for annotations. In total, 7,081 unigenes and 14,099 unigenes were functionally classified into 25 COG categories and 240 KEGG pathways, respectively. Furthermore, 20,216 unigenes were grouped into 48 sub-categories belonging to 3 main Gene Ontology (GO) categories (ontologies). Using the transcriptome data as references, we analyzed the differential gene expressions between a soybean oil-fed group (SOF) and a lard oil-fed group (LOF), compared to the negative control group (NC), using the DGE approach. We finally obtained 1,566 differentially expressed genes in SOF/NC, and 1,099 genes in LOF/NC. For further analysis, GO and KEGG functional enrichment were performed on all differentially expressed genes, and a group of differentially expressed candidate genes related to lipometabolism were identified. CONCLUSIONS/SIGNIFICANCE: This study provides a global survey of C. megacephala and provides the basis for further research on the functional genomics of this insect.

Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR.

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 log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). 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.