The tobacco budworm, Helicoverpa assulta, is a freeze-susceptible species that overwinters in temperate zones with pupa diapause. A rapid cold hardening (RCH) and supercooling capacity usually play crucial roles in survival during the overwintering period. This study is performed to identify a cryoprotectant as a RCH factor in H. assulta. Pre-exposure of H. assulta larvae to 4°C significantly increased survival at -10°C in all developmental stages from egg to adult. RCH was dependent on the duration of the pre-exposure period. RCH also significantly enhanced the supercooling capacity. Cryoprotectant analysis using HPLC showed that the preexposure treatment allowed the larvae to accumulate glycerol in the hemolymph. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol 3-phosphatase (G3P), that involing in glycerol biosynthesis were identified from the transcriptome of H. assulta 4th instar larvae. From the result of transcriptome, the expressions of GPDH and G3P were relatively increased when compared to that of the control, suggesting that these genes contribute to overwintering and biosynthesis of cryoprotectant.

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host

Cotesia plutellae as an endoparasitoid wasp is the natural enemy against the diamondback moth, Plutella xylostella. For successful parasitism of the offspring, the female parasitoid always injects venom with other parasitic factors such as polydnavirus, teratocytes and ovary proteins. These venom products prevent their eggs from host immune system as well as modulate their host. To identify of constituents of venom in relation of host-parasitoid interaction, total RNA of the venom gland were extracted and analyzed with RNA-seq. RNA-seq reveals that 5.5 Gbp were read and approximately 3322 transcripts were identified. The transcripts related to the biological process, cellular components and molecular functions occupied about 20%, 15% and 18%, respectively. Among these transcripts, 301 genes were related to the immune activities such as calreticulin, defensing, phosphatases and serpin etc. These results suggest that some proteins exhibiting biological function related to the immune response can be contribute to the development of wasp in the host.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors such as venom proteins, teratocytes and polydnavirus. In order to identify immune suppressive factors from teratocytes, we collected the supernatant from serum-free culture media of teratocytes. Concentration of secreted proteins from teratocytes was successfully performed with using centricons among tested methods such proteins precipitation and electrophoresed in sodium dodecyl sulfate polyacrylamide gel. The gel slices were directly digested with trypsin using in-gel digestion method and analyzed via LC-Ms-Ms. Molecular weight of peptide fragments were compared with protein database predicted by full-genome sequences of C. plutellae. We identified two immune responsive proteins, which are calreticulin, host cellular response-related gene and neprilysin 2, immune regulator. This result suggests that host immune response is suppressed or regulated by the immune suppressive factors of teratocytes.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors. In order to analyze the gene expression in teratocytes, total RNAs were isolated and genes expressed in the teratocyte were sequenced by Illumina HiSeq2000 RNASeq analysis. The information on RNA sequences was assembled by Trinity and contigs were annotated by Blast analysis. The levels of gene expression were calculated by FPKM. Approximately, 6.3 Gbs were obtained and 34,686 contigs were found and annotated. Forty two percent of contigs were homologous to previously reported genes and classified by gene ontologies: the highly abundant components are metabolic process, biological regulation and cellular process in biological function; binding, catalytic activity and transporter activity in molecular function; cell part, membrane part and organelle in cellular function, respectively. In addition, some teratocyte transcripts of C. plutellae are related to host regulation such as immunosuppression and nutrition: Ankyrin repeat proteins, Serpin, protease, lipase, chitinase and scavenger receptor.

Currently, honeybee colonies are not stable and suffer from the infection of pathogens, affecting the pollination. For the alternatives to this difficulty, Bombus terrestris has been imported and used for pollination in agricultural fields. Although imported insects for pollination are very useful, the potential risk exposing to novel pathogens has been raised. To assess the risk primarily, we designed and synthesized PCR primers for detection of pathogens and parasites in B. terrestris. The samples were obtained from companies importing B. terrestris or field collections and genomic DNAs not showing physical shearing were purified. PCR for detection of pathogen- or parasite-specific gene revealed several DNA fragments were amplified in expected molecular size including Kashmir Bee Virus, Varroa jacobsoni, V. rindereri, Acarapis woodi and Aspergillus flavus. These amplified DNA fragments are in the process of cloning for DNA sequencing to confirm the target gene amplification. We also have plans to optimize the PCR conditions for each amplified target gene and try to develop biomarkers for diagnosis.