Analysis of the Gynaephora qinghaiensis pupae immune transcriptome in response to parasitization by Thektogaster sp.

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.

Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau.

Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)-precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui.

1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris in water-boiled and 50% ethanol-soaked extracts.

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.

Transcriptome characterization and gene expression analysis related to chemoreception in Trichogramma chilonis, an egg parasitoid.

Chemoreception is critical for the survival of insects. Insects have a variety of behavioral responses, such as mating, host searching and ovipositing, in response to different odor signals detected in their living environment. Trichogramma chilonis, an egg parasitoid, acts as an efficient and effective biocontrol reagent for many agricultural and forestry insect pests in many parts of China. However, little is known about the molecular mechanism of the olfaction-evoked behavior in T. chilonis. In the present study, we conducted transcriptome profiling analysis of T. chilonis based on the Illumina high-throughput sequencing platform in order to explore differences of chemoreception between male and female T. chilonis. In this study, a total of 85 chemosensory genes were identified from transcriptomic data, including 45 odorant receptors (ORs), 22 odorant binding proteins (OBPs), 14 ionotropic receptors (IRs), 2 sensory neuron membrane proteins (SNMPs) and 2 chemosensory proteins (CSPs). From the analysis of the transcriptome, most of the candidate olfactory genes had similar expression levels in males and females, including a few OR and OBP genes (TchiOR38, TchiOR39, TchiOR40, TchiOR41, TchiOR42, TchiOR43, TchiOR44, TchiOR45, TchiOBP1, TchiOBP4, TchiOBP10, TchiOBP12, TchiOBP18 and TchiOBP19) which showed male-biased expression. Some annotated unigenes were chosen randomly to have qRT-PCR, which verified the correctness of analysis of transcriptome in T. chilonis. This is the first study to obtain and identify candidate genes related to chemoreception in T. chilonis. Our work lays a solid foundation for related future research on the chemosensory system of T. chilonis at the molecular level and helps advance the use of T. chilonis as biological control agents.

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.

Detection of Ophiocordyceps sinensis in the roots of plants in alpine meadows by nested-touchdown polymerase chain reaction.

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.

Food utilization and growth of cutworm Spodoptera litura Fabricius larvae exposed to nickel, and its effect on reproductive potential.

Food utilization and growth of the 5th and 6th instar Spodoptera litura Fabricius larvae, and its effect on reproduction potential was evaluated by feeding larvae diets with different doses of Ni for 3 generations. Dose-dependent relationships between Ni levels and food consumption and growth were variable with different larval developmental period and Ni exposure duration. RCR, AD and RGR of the 6th instar larvae were much more affected by Ni exposure than those of 5th instar larvae, and the effects were strongest in the 3rd generation. It was found that RCR was significantly stimulated after 1 and 20 mg kg(-1) Ni exposure, while AD was significantly inhibited after 1, 5, 10 and 40 mg kg(-1) Ni exposure. However, lower levels of Ni (≤5 mg kg(-1)) significantly increased and higher levels of Ni (≥10 mg kg(-1)) significantly decreased RGR. In 3 successive generations, 10 mg kg(-1) Ni significantly increased the ECI and ECD of the 5th instar larvae, and 5 mg kg(-1) Ni significantly increased the ECD of the 6th instar larvae. However, ECD were all significantly inhibited with 20 mg kg(-1) Ni exposure. Results also revealed that durations of larvae were shortened at low levels of Ni, but extended at high levels of Ni. Fecundity was inhibited by the highest Ni doses in each generation, while improved by low Ni doses in the 3rd generation. Hatching rates in all treatments were significantly decreased in a Ni dose-dependent manner. Study indicated that effects of Ni on these parameters were predominant with the increasing Ni exposure period.

The effect of dietary nickel on the immune responses of Spodoptera litura Fabricius larvae.

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.