[Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)].

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/ß-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Fluorescent Nanoparticle-RNAi-Mediated Silencing of Sterol Carrier Protein-2 Gene Expression Suppresses the Growth, Development, and Reproduction of Helicoverpa armigera.

Helicoverpa armigera is a polyphagous destructive lepidopteran pest with strong Bacillus thuringiensis (Bt) resistance. Cholesterol, a vital component for insect growth, can only be obtained from food, and its transfer and metabolism are regulated by sterol carrier protein-2 (SCP-2). This study examined whether H. armigera SCP-2 (HaSCP-2) gene expression, involved in cholesterol absorption, can be silenced by nanocarrier fluorescent nanoparticle-RNA interference (FNP-RNAi) by larval feeding and whether the silencing affected H. armigera development. Fluorescence microscopy showed that nanoparticle-siRNA was distributed in Ha cells and the larval midgut. FNP-HaSCP-2 siRNA suppressed HaSCP-2 expression by 52.5% in H.armigera Ha cells. FNP can effectively help deliver siRNA into cells, protect siRNA, and is not affected by serum. FNP-siRNA in vivo biological assays showed that HaSCP-2 transcript levels were inhibited by 70.19%, 68.16%, and 67.66% in 3rd, 4th, and 5th instar larvae, leading to a decrease in the cholesterol level in the larval and prepupal fatbodies. The pupation rate and adult emergence were reduced to 26.0% and 56.52%, respectively. This study demonstrated that FNP could deliver siRNA to cells and improve siRNA knockdown efficiency. HaSCP-2 knockdown by FNP-siRNA in vivo hindered H. armigera growth and development. FNP could enhance RNAi efficiency to achieve pest control by SCP-2-targeted FNP-RNAi.

Transcriptomic profiling of the flower scent biosynthesis pathway of Cymbidium faberi Rolfe and functional characterization of its jasmonic acid carboxyl methyltransferase gene.

BACKGROUND: Cymbidium faberi, one of the most famous oriental orchids, has a distinct flower scent, which increases its economic value. However, the molecular mechanism of the flower scent biosynthesis was unclear prior to this study. Methyl jasmonate (MeJA) is one of the main volatile organic compounds (VOC) produced by the flowers of C. faberi. In this study, unigene 79,363 from comparative transcriptome analysis was selected for further investigation. RESULTS: A transcriptome comparison between blooming and withered flowers of C. faberi yielded a total of 9409 differentially expressed genes (DEGs), 558 of which were assigned to 258 pathways. The top ten pathways included α-linolenic acid metabolism, pyruvate metabolism and fatty acid degradation, which contributed to the conversion of α-linolenic acid to MeJA. One of the DEGs, jasmonic acid carboxyl methyltransferase (CfJMT, Unigene 79,363) was highly expressed in the blooming flower of C. faberi, but was barely detected in leaves and roots. Although the ectopic expression of CfJMT in tomato could not increase the MeJA content, the expression levels of endogenous MeJA biosynthesis genes were influenced, especially in the wound treatment, indicating that CfJMT may participate in the response to abiotic stresses. CONCLUSION: This study provides a basis for elucidating the molecular mechanism of flower scent biosynthesis in C. faberi, which is beneficial for the genetically informed breeding of new cultivars of the economically valuable oriental orchids.

Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells.

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.

NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm.

The cotton bollworm, Helicoverpa armigera, has developed strong resistance to many insecticides. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new target. In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. HaSCP-2 composed of five α-helices and four stranded ß-sheets. The folds of α-helices and ß-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. Several sterols and fatty acids could interact with HaSCP-2 via important hydrophobic sites, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51, F53, F89, F110, I117 and Q131 may be the key functional sites. HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo. Our study describes for the first time a NMR structure of SCP-2 in lepidopteran H. armigera and reveals its important function in cholesterol uptake, which facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.

Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae).

The midgut of lepidopteran larvae serves as a target for many pathogens such as Bacillus thuringiensis (Bt). Cell lines originating from midgut tissues will be very helpful tools in many research fields. However, to date, no Bt-susceptible midgut-derived cell lines are available. Here, we reported that a novel cell line, designated as HNU-Ha-MG1, was established from midgut tissue of the fourth instar larvae of Helicoverpa armigera. This cell line grew well in Grace's insect cell culture medium supplemented with 10-15% fetal bovine serum. The shape of the most cells was round or polygonal, and some tended to aggregate to form multiple cell masses. The size of the cells was 13.8 ± 1.8 µm in diameter, and the maximum density reached (2.40 ± 0.15) × 10(6) cells/ml. The population doubling time during logarithmic growth phase was 58.6 ± 7.0 h at 28°C. The number of chromosomes was about 90-130, which exhibited typical chromosome characteristics of lepidopteran cell lines. The patterns of random amplified polymorphic DNA of the cell line were different from those of Sl-HP and Hi5 cell lines which were frequently used in our laboratory. 20-Hydroxyecdysterone induced apoptosis in a very small part of cells at 2 µg/ml but did not affect expression of autophagy-related protein 8 (Atg8) and its lipidation at 36 h post-treatment. The cell line was permissive to Autographa californica nuclear polyhedrosis virus (AcMNPV) and H. armigera nuclear polyhedrosis virus (HaSNPV). This cell line was found to be susceptible to activated Cry1C at the final concentration of 0.5-1.0 µg/ml but not to the activated Cry1Ac.

Endogenous expression of a Bt toxin receptor in the Cry1Ac-susceptible insect cell line and its synergistic effect with cadherin on cytotoxicity of activated Cry1Ac.

Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action.

Distribution, cleavage and lipidation of Atg8 fusion proteins in Spodoptera litura Sl-HP cells.

Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.

Characterization of Atg8 in lepidopteran insect cells.

Yeast Atg8 and mammalian microtubule-associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. Sequence analysis reveals that Atg8 proteins are highly conserved in lepidopteran species. The abundance of endogeous Atg8 and the ratios of Atg8 conjugation to phosphatidylethanolamine (Atg8-PE)/Atg8 are different among several lepidopteran cell lines and different tissues of Helicoverpa armigera larvae. Both the density of fluorescent pre-autophagosomal structures with GFP-Ha Atg8 and the abundance of Atg6 are positively correlated with levels of Atg8-PE in different cell lines. The mutant GFP-Atg8(G116A) has lost the function in punctual formation, suggesting that G116 is important for autophagy. Exogenous factors have significant influences on the conversion of Atg8 in lepidopteran cells. Bacillus thuringiensis enhances the degradation of Atg8 in Spodoptera litura Sl-HP cells. Atg8-PE degrades gradually with extension of amino acid starvation, and bafilomycin A1 can block the decrease through the inhibition of autophagosome fusion with lysosome. Interestingly, high pH is more effective than amino acid starvation in Bombyx mori Bme cells to induce the conversion of BmAtg8 to BmAgt8-PE. Change of the quality of fetal bovine serum in the culture medium results in alteration of the ratio of Atg8-PE/Atg8 in some lepidopteran cell lines.

Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin.

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.

THAP and ATF-2 regulated sterol carrier protein-2 promoter activities in the larval midgut of the yellow fever mosquito, Aedes aegypti.

Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream -1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the -1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the -1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled -1.6/-1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between -1.6 to -1.3 kb 5' upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression.

The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.

Phylogenetic diversity of nitrogen fixation genes in the intestinal tract of Reticulitermes chinensis Snyder.

Wood-feeding termites live on cellulolytic materials that typically lack of nitrogen sources. It was reported that symbiotic microbes play important roles in the maintenance of a normal nitrogen contents in termite by different metabolisms including nitrogen fixation. In this study, the diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of Reticulitermes chinensis Snyder was investigated with culture independent method. Fragments of the nifH genes, which encode dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut with four sets of primers corresponding to the conserved regions of the genes. Clones were randomly selected and analyzed by RFLP. Sequence analysis revealed that a large number of nifH sequences retrieved from the termite gut were most closely related to strict anaerobic bacteria such as clostridia and spirochetes, some of the others were affiliated with proteobacteria, bacteroides, or methanogenic archaea. The results showed that there was a remarkable diversity of nitrogenase genes in the gut of Reticulitermes chinensis Snyder.

Baculovirus infection triggers a shift from amino acid starvation-induced autophagy to apoptosis.

Autophagy plays a central role in regulating important cellular functions such as cell survival during starvation and control of infectious pathogens. On the other hand, many pathogens have evolved mechanisms of inhibition of autophagy such as blockage of the formation of autophagosomes or the fusion of autophagosomes with lysosomes. Baculoviruses are important insect pathogens for pest control, and autophagy activity increases significantly during insect metamorphosis. However, it is not clear whether baculovirus infection has effects on the increased autophagy. In the present study, we investigated the effects of the Autographa californica nucleopolyhedrovirus (AcMNPV) infection on autophagy in SL-HP cell line from Spodoptera litura induced under amino acid deprivation. The results revealed that AcMNPV infection did not inhibit autophagy but triggered apoptosis under starvation pressure. In the early stage of infection under starvation, mitochondrial dysfunction was detected, suggesting the organelles might be involved in cell apoptosis. The semi-quantitative PCR assay revealed that the expression of both p35 and ie-1 genes of AcMNPV had no significant difference between the starved and unstarved SL-HP cells. The western blot analysis showed that no cleavage of endogenous Atg6 occurred during the process of apoptosis in SL-HP cells. These data demonstrated that some permissive insect cells may defend baculovirus infection via apoptosis under starvation and apoptosis is independent of the cleavage of Atg6 in SL-HP cells.

Deinococcus reticulitermitis sp. nov., isolated from a termite gut.

Bacterial strain TM-1(T) was isolated from the gut of a wood-feeding termite, Reticulitermes chinensis Snyder. Cells of strain TM-1(T) were Gram-negative, spherical (1.0-2.0 µm in diameter), non-motile, non-sporulating and red-pigmented. Strain TM-1(T) was resistant to UV radiation, showing 34% survival after exposure to UV light at a dose of 100 J m(-2). Growth occurred at 20-40 °C (optimum, 30 °C), at pH 6.0-10.0 (optimum, pH 6.0-7.0), and in the presence of 0-1% (w/v) NaCl (optimum, 0-0.4%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TM-1(T) was related to members of the genus Deinococcus, with sequence similarities ranging from 87.0 to 94.0%. The peptidoglycan of strain TM-1(T) contained ornithine, alanine, glycine and glutamic acid. The most abundant cellular fatty acids of strain TM-1(T) were summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c; 22.3%) and C(16:0) (37.5%). MK-8 was the predominant quinone. The polar lipid profile contained three glycophospholipids, six glycolipids, one aminolipid and three unknown lipids. DNA of the type strain had a G+C content of 65.6 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented, strain TM-1(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus reticulitermitis sp. nov. is proposed, with TM-1(T) (=CGMCC 1.10218(T)=NBRC 106334(T)) as the type strain.

Responses of two insect cell lines to starvation: autophagy prevents them from undergoing apoptosis and necrosis, respectively.

The responses of insect cells to starvation and the characteristics of cell death after the depletion of nutrients remain largely unknown. In the present study, we investigated autophagy, apoptosis and necrosis in two Lepidoptera insect cell lines in response to amino acid starvation. Our data demonstrated that starvation induced a significant increase in autophagy in Spodoptera litura SL-ZSU-1 cells, and cell apoptosis followed autophagy after starvation of more than 48h. However, at an early stage of starvation, inhibition of autophagy with 3-MA rapidly triggered apoptosis of SL-ZSU-1 cells, suggesting autophagy inhibits cell apoptosis. By contrast, Bombyx mori SPC Bm36 cells died by a non-apoptotic pathway if the starvation was prolonged for more than 48 h. At the early stage of starvation, inhibition of autophagy with 3-MA did not trigger apoptosis in Bm36 cells, but resulted in necrotic-like cell death. Under starvation pressure, autophagy in SL-ZSU-1 cells was much more active than in Bm36 cells. The activity of caspase-9-like in apoptotic SL-ZSU-1 cells also was much higher than in apoptotic Bm36 cells. RT-PCR analyses revealed that transcriptional levels of saposin-like (Bm109) and Atg6 were undetectable in Bm36 cells, but expression level of saposin-like in SL-ZSU-1 was high. Expression of Atg6 in SL-ZSU-1 cells was not analyzed because its sequence was unknown. These data indicate that autophagy prevents Lepidoptera insect cells from death at an early stage of starvation, but prolonged starvation results in cell death. The pathways of cell death might be dependent on the abundance of caspase-9-like, saposin-like and Atg6.

Highly passage of Spodoptera litura cell line causes its permissiveness to baculovirus infection.

It is well known that the characteristics of cell lines possibly alter when cell lines are at high-passage number because of the environmental selection. We do not know whether non-permissive or low-permissive cell lines could become permissive or more permissive to virus infection after over-high passage. In the present studies, the alteration of the permissiveness of Spodoptera litura cell line Sl-zsu-1 to three baculovirus infection was investigated after over-high passage, and the possible mechanisms are also investigated. Vigorous apoptosis in Sl-zsu-1 cells was induced by both the recombinant Autographa californica multiple nucleopolyhedrovirus AcMNPV-GFP-actin and the celery looper Anagrapha falcifera multiple nucleopolyhedrovirus AfMNPV, suggesting the replication of the two viruses was blocked by apoptosis. However, the cells infected by S. litura multicapsid nucleopolyhedrovirus SpltMNPV did not undergo apoptosis, but the SpltMNPV titre of the supernatant was not detectable, suggesting this cell line was low-permissive for this virus infection and other factor(s) involved in blockage of the virus replication except apoptosis. However, when Sl-zsu-1 cells had been subcultured continuously for more than 4 years (high-passage cell), which was named as Sl-HP cell line afterwards, no significant apoptosis was induced by the three baculovirus in Sl-HP cells, and many replicated virions or nucleocapsids were observed in the cells. But the permissiveness of Sl-HP cells to the three viruses was very different according to the titre of viruses in the cell cultures. Interestingly, the DNA extracted from SpltMNPV could induce vigorous apoptosis of Sl-HP cells. Altogether, Sl-zsu-1 cell line becomes more permissive to baculovirus infection after over-high passage and multiple paths can block the baculovirus infectivity.

Influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV) in insect Spodoptera litura cells.

We investigated the influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV). Microscopic observation revealed that infection of SL-1 cells with AfMNPV resulted in apoptosis, displaying apoptotic bodies in fluorescent-stained nuclei of AfMNPV-infected SL-1cells. Western blot analysis demonstrated that AfMNPV-induced apoptosis in insect SL-1 cells was significantly inhibited by cyclosporin A which blocked a translocation of cytochrome c from the mitochondria to the cytosol. As determined by using AC-DEVD-AFC as substrate, the activity of caspase-3 in AfMNPV-induced cells was detected as early as 4h post infection, gradually increased with time extension, and reached a highest level after 16h of infection. However, activity of caspase-3 in apoptotic cells decreased in the presence of cyclosporin A (30microM), indicating that activation of caspase-3 in SfaMNPV-induced cells was dependent on the release of cytochrome c from the mitochondria. In addition, cyclosporin A could markedly inhibit mitochondrial transmembrane potential (DeltaPsim) disruption in undergoing apoptotic cells. These data indicate that cytochrome c plays a key role in AfMNPV-induced apoptosis in S. litura cells and may be required for caspase activation during the induction of apoptosis.

Optical brightener M2R destroys the peritrophic membrane of Spodoptera exigua (Lepidoptera: Noctuidae) larvae.

Observations under an environmental scanning electron microscope showed that the peritrophic membrane (PM) of Spodoptera exigua (Hübner) was smooth without pores, but there were many pores and slits in the PM of larvae fed with 10 g L(-1) optical brightener Calcofluor white M2R. After incubation of S. exigua PM in vitro with 10 g L(-1) M2R, a significant amount of proteins was released from the PM structure. M2R disrupted the structure of larval PM, resulting in greatly increased permeability and increased larval susceptibility to Syngrapha falcifera multiple nucleopolyhedrovirus infection. Continuous feeding of larvae on a diet treated with a 10 g L(-1) M2R solution significantly retarded larval development and resulted in high mortality. The destructive effect of M2R on PM was transient and reversible, depending on the presence of M2R within the midgut.

Influence of glucose starvation on the pathway of death in insect cell line Sl: apoptosis follows autophagy.

The relation between autophagy and apoptosis has not been clearly elucidated. Here, we reported that apoptosis followed autophagy in insect Spodoptera litura cells (Sl) undergoing glucose starvation. Sl cells have been adapted to Leibovitz-15 medium supplemented with glucose (1.0 g/l) and 5% fetal bovine serum (FBS), used for mammalian cell cultures. If glucose (1 g/l) or glutamine (1.6 g/l) had not been supplemented in L-15 medium with 5% FBS, Sl cells began to form many vacuoles and these vacuoles gradually enlarged in the cytoplasm, which were autophagic vacuoles. However, these large vacuoles began to disappear gradually after 48 h of glucose starvation, accompanied with remarkable apoptosis without apoptotic bodies, which was demonstrated by DNA fragmentation and activation of caspase-3-like. During glucose starvation, Sl cell ATP concentrations gradually decreased. Interestingly, if the conditioned L-15 medium without glucose was replaced with fresh L-15 medium supplemented with glucose or glutamine after the cultures had been starved seriously for 48 h or longer, the formation of apoptotic bodies was initiated. These data suggested that the partial depletion of cell ATP triggered apoptosis following autophagy in glucose-starved Sl cells and the formation of apoptotic bodies required higher level of ATP than DNA fragmentation and activation of caspase-3-like activity. Additionally, the disappearance of autophagic vacuoles, negative staining of neutral red, green staining of acridine orange and diffusion of acid phosphatase activity in Sl cells at the late stage of starvation (over 48 h) suggested that the dysfunction of lysosome was more likely to involve in apoptosis. The facts that Actinomycin D-induced apoptosis was partially inhibited and cyclosporin A, blocking the opening of mitochondrial permeability transition (MPT) pores, inhibited partially apoptosis in glucose-starved Sl cells, suggested the pathway of glucose starvation-induced apoptosis seemed to be different from that induced by actinomycin D and the opening of MPT pores on mitochondria probably involved in apoptosis triggered by glucose starvation, respectively.