Hong-Lian Shao

Establishment of a New Cell Line from Lepidopteran Epidermis and Hormonal Regulation on the Genes

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

Molecular cloning and expression analysis of signal transducer and activator of transcription (STAT) from the Chinese white shrimp Fenneropenaeus chinensis

Innate immunity is the first line of defense by a host against invading pathogens. Several signaling pathways participate in the immune response, one of which is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Various evidences have been provided to suggest that the JAK/STAT pathway is involved in both antibacterial and antiviral immunities. In this study, the full-length cDNA and gene sequence of STAT (designated as FcSTAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis. Phylogenetic analysis reveals that the FcSTAT is clustered with STAT5s and STAT6s from vertebrates and STATs from invertebrates. Quantitative real-time PCR exhibited that the FcSTAT had a wide distribution in all detected tissues and developmental stages. Time course analysis of the transcription level after WSSV challenge showed a noticeably early up-regulation of FcSTAT in hemocytes, hepatopancreas, and intestines. The expression levels of FcSTAT increased corresponding to Vibrio anguillarum stimulation in both hemocytes and hepatopancreas as well. All these imply that the JAK/STAT pathway participates in the immune response against bacteria and virus in F. chinensis.

β-thymosin is upregulated by the steroid hormone 20-hydroxyecdysone and microorganisms

Thymosins have diverse biological activities including actin‐sequestering and tissue repair in vertebrates, however, there is little information about the function of thymosins in invertebrates. We isolated a β‐thymosin gene in Helicoverpa armigera. It has two transcript variants, HaTHY1 and HaTHY2, encoding 19.0 kDa and 14.5 kDa peptides, respectively. HaTHY1 was mainly transcribed in the integument and midgut, while HaTHY2 was principally presented in the fat body and haemocytes. The transcript levels of HaTHY2 showed some fluctuation; there was an obvious increase at the metamorphic stage in the integument or fat body. HaTHY was able to be upregulated by 20‐hydroxyecdysone or by bacterial and viral challenge. These data suggest that HaTHY is upregulated by the steroid hormone and by responses to microorganism infection.

Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

BackgroundAdenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms.ResultsThis study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown.ConclusionThese results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development.

SUPPRESSION OF AcMNPV REPLICATION BY ADF AND THYMOSIN PROTEIN UP-REGULATION IN A NEW TESTIS CELL LINE, Ha-shl-t

Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha-shl-t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up-regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F-actin. Silencing thymosin or adf or treating cells with the F-actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F-actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha-shl-t cells utilize F-actin depolymerization to suppress AcMNPV replication by up-regulating thymosin and adf. We propose Ha-shl-t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally.

Reconstruction of AcMNPV with Helicoverpa hormone receptor 3 and its effect on the Helicoverpa larvae

Abstract Helicoverpa hormone receptor 3 (HHR3) is a transcription factor involved in larval molting regulation. To investigate its function and the possibility to improve the insecticidal activity of AcMNPV, HHR3 was expressed in Bac-to-Bac system and an AcMNPV-GFP-HHR3-Polh+ virus was constructed after recovery of the polyhedrin gene. HHR3 expression in Sf21 cells was confirmed by immunofluorescence microscopy. A red color identifying HHR3 was observed using fluorescence microscopy; this confirmed that HHR3 was expressed in Sf21 cells by the introduction of the recombinant virus AcMNPV-GFP-HHR3-Polh+. The AcMNPV-GFP-HHR3-Polh+ infected the hemocytes of H. armigera larvae by hemocoele injection or per oral ingestion. Results showed that AcMNPV-GFP-HHR3-Polh+ resulted in incomplete ecdysis, slower growth and lower median survival time on the Helicoverpa larvae. This recombinant virus may aid in the development of an insecticide that can be used in the field.

Purification and characterisation of an inhibitor of a cathepsin B-like proteinase from sunflower seed

Cathepsin B is a vitally important enzyme in various physiological processes and in tumor invasion and metastasis. A cathepsin B inhibitor, HCB-SunI, was identified and purified from sunflower seeds, Helianthus annuus, using ammonium sulfate precipitation and two steps of conventional chromatography. The molecular mass of HCB-SunI was estimated to be 12 kDa by SDS-PAGE and 12.32 kDa by MALDI TOF MS. Its N-terminal amino acid sequence was determined to be: PYGGGGTESG. HCB-SunI not only inhibited Helicoverpa cathepsin B (HCB) but also decreased the growth of HeLa and glioma cells by 7 ∼ 27% and 6 ∼ 22%, respectively, when the cells were grown in a final concentration of 0.002 ∼ 0.008 μM inhibitor.