Yuzhu Wang

A cell strain cloned from Spodoptera exigua cell line (IOZCAS-Spex-II) highly susceptible to S. exigua nucleopolyhedrovirus infection

A cell strain (IOZCAS-Spex-II-A) cloned from IOZCAS-Spex-II, a cell line established from the fat body of Spodoptera exigua (Lepidoptera: Noctuidae) larva, was characterized, and its capability to produce S. exigua nucleopolyhedrovirus was high with infection rate exceeding 90% compared with its parental cell line IOZCAS-Spex-II that scored only 50%. Growth curve of budded virus (BV) in the strain was analyzed and the titer of BV reached the highest of 3.7u2009×u2009104 pfu/mL by 96xa0h after inoculation. Concentration of occlusion bodies (OBs) produced by the cloned cell strain (IOZCAS-Spex-II-A) was 7.1u2009×u2009107 OBs/mL, while the parental cell line produced 2.4u2009×u2009107 OBs/mL. The average yield of the virus was 176 OBs/cell of IOZCAS-Spex-II-A compared with 211 OBs/cell that of the parental cell line. Significant differences were observed in virus production, growth characters, cell shape, between the parental cell line, and its clone. The cell lines (IOZCAS-Spex-II and IOZCAS-Spex-II-A) were also susceptible to Autographa californica multiple nucleopolyhedrovirus infection. In addition, they were characterized with regard to their growth rates and DNA amplification fingerprinting technique employing polymerase chain reaction.

A NEW CELL LINE FROM LARVAL FAT BODIES OF THE BOLLWORM, HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE)

SummaryA new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.

Two new cell lines originated from the embryos of Clostera anachoreta (Lepidoptera: Notodontidae): characterization and susceptibility to baculoviruses

Two cell lines designated CAF-Clan I and CAF-Clan II have been established from embryos of Clostera anachoreta (Lepidoptera: Notodontidae) in TNM-FH medium containing 10% inactivated fetal bovine serum. CAF-Clan I consists of a mixture of three cell types: spherical cells, spindle-shaped cells, and giant cells. Most of the cultured cells formed a suspension in the medium and were subcultured more than 60 passages. CAF-Clan II mainly consists of spindle-shaped and spherical cells which attached to the culture surface and have undergone more than 40 passages. The cell population doubling time at 27°C of CAF-Clan I at passage 22 and CAF-Clan II at passage 24 was about 68.5 and 38.2xa0h, respectively. The chromosome number of both cell lines at passage 15 varied from 62 to 100 in the majority of cells, though a few cells exceeded 260 (nu2009=u200930). DNA amplification fingerprinting–polymerase chain reaction analysis confirmed that the origination of the two cell lines was C. anachoreta. The susceptibility of the cell lines to baculoviruses was tested. The results showed that CAF-Clan II was susceptible to infection of Autographa californica nucleopolyhedrovirus (AcMNPV) and Ecotropis oblique nucleopolyhedrovirus (EoNPV). Occlusion bodies (OBs) production was 129u2009±u20094 OBs/cell and 124u2009±u200915 OBs/cell for AcMNPV and EoNPV, respectively. CAF-Clan I was less susceptible to AcMNPV compared with CAF-Clan II, while non-permissive to EoNPV.

Metabolic profiling of somatic tissues from Monochamus alternatus (Coleoptera: Cerambycidae) reveals effects of irradiation on metabolism.

A high-level of sexual sterility is of importance for the sterile insect technique (SIT). However, the use of high-dose-intensity gamma radiation to induce sterility has negative impacts not only on reproductive cells but also on somatic cells. In this study, we investigated the metabolite differences in somatic tissues between non-irradiated, 20-Gy-irradiated, and 40-Gy-irradiated male Monochamus alternatus, an important vector of the pathogenic nematode, Bursaphelenchus xylophilus, which kills Asian pines. The results showed that metabolite levels changed moderately in the 20-Gy samples but were markedly altered in the 40-Gy samples compared with the non-irradiated samples. Twenty-six and 53 metabolites were disturbed by 20-Gy and 40-Gy radiation, respectively. Thirty-six metabolites were found to be markedly altered in the 40-Gy samples but were not changed significantly in the 20-Gy samples. The comprehensive metabolomic disorders induced by 40-Gy radiation dysregulated six metabolic pathways involved in the life process. The findings presented in this manuscript will contribute to our knowledge of the characteristic metabolic changes associated with gamma-radiation-induced damage to somatic cells and will allow for better exploration of the SIT for the control of this target pest.

Rapid molecular detection of Esteya vermicola based on specific primers and the FTA-DNA extraction method

Esteya vermicola is an endoparasitic fungus of the pinewood nematode and thus has great biocontrol value. At present, the detection of this fungus is still based on microscopic observations and morphological identification, and the sampling is notably inconvenient and inefficient. In the present study, a pair of specific primers (upstream primer, 5′-GTGCCTCTACCAAGACTCGC-3′; downstream primer, 5′-CGCCAAATGTCAAGATCCGC-3′) was designed to detect E. vermicola. The analysis of the PCR amplification and the agarose gel electrophoresis results led to the establishment of a new method for the detection of E. vermicola through the presence of a 176-bp specific fragment. In addition, the use of a FTA-DNA direct extraction method for the detection of E. vermicola was explored. The results suggest that the proposed method can be effectively used for the rapid detection of E. vermicola and may provide important technical support for follow-up studies of the fungus in field experiments.

Characterization of a novel chitinase, DkChi, from Dendrolimus kikuchii nucleopolyhedrovirus

Dendrolimus kikuchii Matsumura nucleopolyhedrovirus (DkNPV) is a novel nucleopolyhedrovirus strain that has exhibited high potential as biological control agent against D. kikuchii. In this work, a 1755-bp DkChi gene with sequence homology to a chitinase gene was cloned from the genomic DNA of DkNPV using a DNA fragment library. The DkChi gene, encoding 558 residues protein with a predicted mass of 61.6 kDa, was expressed at high levels in Escherichia coli and purified by affinity chromatography. We confirmed that the prepared protein was the DkChi protein by mass spectrometry analysis. Enzyme activity analysis showed that DkChi had both endo- and exo-chitinase activities. Interestingly, the DkChi protein displayed a strong insecticidal activity against Spodoptera exigua, Hyphantria cunea, Helicoverpa armigera and Lymantria dispar. The results suggest that DkChi is a good candidate protein for significantly contributing to pest control.

Transgenic expression of Dendrolimus kikuchii nucleopolyhedrovirus enhancin in tobacco enhances the mortality and affects the development of Spodoptera exigua larvae

ABSTRACT A novel 2551-bp enhancin gene designated En-Dk was isolated from the Dendrolimus kikuchii nucleopolyhedrovirus genome. Sequence analysis revealed that En-Dk encodes a polypeptide chain of 792 amino acid residues containing a conserved enhancin domain and an α-helical transmembrane domain, but no signal peptide. Compared with wild-type tobacco (Nicotiana tabacum L.), transgenic tobacco showed significant phenotypic changes following En-Dk overexpression with slower growth and delayed initial flowering. An interesting finding is that the transgenic tobacco exerted a toxic effect on newly hatched Spodoptera exigua larvae, with a significant inhibitory effect on third-instar larvae in terms of body weight, pupal weight, pupation and the emergence process.

Infection of a nucleopolyhedrovirus to Neodiprion zhejiangenis Zhou & Xiao (Hymenoptera: Diprionidae)

ABSTRACT Neodiprion zhejiangenis Zhou & Xiao 1981 (Hymenoptera: Diprionidae) nuclear polyhedrosis virus (NezhNPV) is a biocontrol factor with high development potential and application prospects. To study the insecticidal activity of NezhNPV against the larvae of N. zhejiangenis, the susceptibility of larvae at different instars to different NezhNPV concentrations was determined based on laboratory bioactivity. At 2.0u2009×u2009107 polyhedron inclusion body (PIB)/mL, the median lethal time required to kill 50% of the instar 2 and 3 larvae was approximately 5 d, whereas the median lethal time of the instar 4 and 5 larvae was 8–9 d. Interestingly, at the NezhNPV concentrations of 2.0u2009×u2009103 and 2.0u2009×u2009104 PIB/mL, the feeding ability of the surviving larvae was decreased by 57.4% and 76.4%, respectively, compared with the controls; the pupal weight at both concentrations was decreased by approximately 27%, and the adult emergence rate was decreased by 27.4% and 50.9%, respectively, with a significantly higher proportion of males than of females. The results showed that younger instar larvae of N. zhejiangenis were more susceptible to NezhNPV infection than older instar larvae. The larval mortality rate was dependent on the larval instar and NezhNPV concentration.