Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration.

Pv11, a cell line derived from the anhydrobiotic insect, Polypedilum vanderplanki, was preserved in a dry form (only 6% residual moisture) at room temperature for up to 251 days and restarted proliferating after rehydration. A previous study already reported survival of Pv11 cells after desiccation, but without subsequent proliferation. Here, the protocol was improved to increase survival and achieve proliferation of Pv11 cells after dry storage. The method basically included preincubation, desiccation and rehydration processes and each step was investigated. So far, preincubation in a 600 mM trehalose solution for 48 h before dehydration was the most favourable preconditioning to achieve successful dry preservation of Pv11 cells, allowing about 16% of survival after rehydration and subsequent cell proliferation. Although the simple air-dry method established for Pv11 cells here was not applicable for successful dry-preservation of other insect cell lines, Pv11 is the first dry-preservable animal cell line and will surely contribute not only to basic but also applied sciences.

Transcriptional regulation of juvenile hormone-mediated induction of Krüppel homolog 1, a repressor of insect metamorphosis.

The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.

Expression of recombinant proteins by BEVS in a macula-like virus-free silkworm cell line.

We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells. These findings indicate that persistent infection with BmMLV has no discernible effect on BmNPV-mediated protein production in B. mori cells.

RNAi suppression of ß-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells.

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific ß-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.

Production of small antibacterial peptides using silkworm-baculovirus protein expression system.

The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human ß-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm-baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

A new continuous cell line from the pest insect, Anomala cuprea (Coleoptera; Scarabaeidae): emergence of contractile cells.

Insect contractile cells frequently appear at an early phase of cell culture, but in most cases, they disappear before a continuous cell line is established, so the cell line ceases to contract. Continuous contractile insect cell lines are currently available from only one species each of Hymenoptera and Diptera. Here, we obtained a new cell line that contracted long after being established as a continuous cell line. The cell line contracted for a short period at an early phase of insect cell culture before a continuous cell line was established, but then did not contract again for several years. After this cell line entered the continuous growth phase, it produced spontaneously contractile tissues for about 4 mo but stopped contracting again. This is the first instance of a cell line that contracted after its establishment as a non-contractile continuous cell line. It is unclear whether the contractile cells survive or die after contraction ceases at an early phase of cell culture, and our results indicate that potential contractile cells survive for years after they stop to contract. The cells of this line sometimes produced complex contractile structures, such as sheet-like tissues. Only a few continuous cell lines have been derived from scarabaeid beetles. The new continuous cell line was derived from the culture of the fat bodies of the scarab beetle Anomala cuprea, which is a pest in the agriculture and forestry of Japan. The population doubling time of the new cell line was 2.5 d and thus it grows very rapidly among coleopteran continuous cell lines. Our new cell line will facilitate research on the physiology and pathology of Coleoptera, including scarab beetles, and may also contribute to research on invertebrate muscles.

Nucleotide sequence and chromosomal localization of the gene for pierisin-1, a DNA ADP-ribosylating protein, in the cabbage butterfly Pieris rapae.

Cabbage butterfly, Pieris rapae, contains a unique DNA ADP-ribosylating protein, pierisin-1, which transfers ADP-ribose moiety of NAD to guanine bases of DNA. Pierisin-like proteins are only distributed in subtribes Pierina, Aporiina and Appiadina of the family Pieridae. In this study, we obtained genomic clones carrying the pierisin-1 gene from adult samples of P. rapae by plaque hybridization. The pierisin-1 gene was found to consist of two exons, 0.1-kb exon 1 and 3.9-kb exon 2, and a 2.3-kb intron. In addition, we could demonstrate that the putative promoter in the about 3-kb upstream region from the transcription start site of the gene include a transcriptional activating motif involved in immune pathways and hormonal regulation. We also examined chromosomal localization of the pierisin-1 gene. Fluorescence in situ hybridization (FISH) analysis using Cy3-labeled pierisin-1 genomic clone demonstrated the localization of the gene near the kinetochore in chromosome 9. Thus, we confirmed that the pierisin-1 gene is located in the genome of P. rapae.

Expression profiling of novel bacteria-induced genes from the silkworm, Bombyx mori.

In this study, we have newly identified three bacteria-induced genes from the silkworm Bombyx mori by quantitative reverse transcriptase-polymerase chain reaction. One of these, eukaryotic initiation factor 4E-1 (eIF4E-1), is assumed to encode an eIF4E family, which plays a role in the initiation of translation as a mRNA cap-binding protein. The second gene is BmFOXG1, belonging to a family of forkhead transcription factors, FOXG1. The third gene is MBF2-related (MBF2-R) whose product has high homology to a co-activator protein MBF2 from B. mori. Although BmFOXG1 was up-regulated in the fat body in response to three kinds of bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, eIF4E-1 and MBF2-R were up-regulated by E. coli and B. subtilis, but not S. aureus, suggesting that bacteria possessing meso-diaminopimelic acid-containing peptidoglycan but not lysine-containing peptidoglycan activate eIF4E-1 and MBF2-R, probably through a conserved immune deficiency pathway. We further profiled the expression of three genes in different tissues and a silkworm cell line, NIAS-Bm-aff3, in response to bacteria, and at different times after bacterial challenge in the fat body.

Cells from an anhydrobiotic chironomid survive almost complete desiccation.

Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation.

shRNA expression plasmids generated by a novel method efficiently induce gene-specific knockdown in a silkworm cell line.

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

Functional characterization of a cactus homolog from the silkworm Bombyx mori.

A cDNA encoding an IkappaB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IkappaB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A glutathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.

Correlation of differential expression of silkworm antimicrobial Peptide genes with different amounts of rel family proteins and their gene transcriptional activity.

In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to kappaB sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.

DNA vector-based RNA interference in cell lines derived from Bombyx mori.

Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

Establishment and characterization of the Bombyx mandarina cell line.

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 degrees C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei's genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.

Inactivation of Bombyx mori macula-like virus under physical conditions.

The Bombyx mori macula-like virus (BmMLV) is a member of the genus Maculavirus, family Tymoviridae, and contains a positive-sense single-stranded RNA genome. Previously, we reported that almost all B. mori-derived cell lines have already been contaminated with BmMLV via an unknown infection route. Since B. mori-derived cell lines are used for the baculovirus expression vector system, the invasion of BmMLV will cause a serious safety risk in the production of recombinant proteins. In this study, to determine the inactivation effectiveness of BmMLV, viruses were treated with various temperatures as well as gamma and ultraviolet (UV) light radiation. After these treatments, the virus solutions were inoculated into BmMLV-free BmVF cells. At 7 days postinoculation, the amount of virus in cells was evaluated by real-time reverse transcription PCR. Regarding heat treatment, conditions under 56°C for 3 h were tolerated, whereas infectivity disappeared after treatment at 75°C for 1 h. Regarding gamma radiation treatment, viruses were relatively stable at 1 kGy; however, their infectivity was entirely eliminated at a dose of 10 kGy. With 254 nm UV-C treatment, viruses were still active at less than 120 mJ/cm(2); however, their infectivity was completely lost at greater than 140 mJ/cm(2) UV-C radiation. These results provide quantitative evidence of the potential for BmMLV inactivation under a variety of physical conditions.

Infection studies of nontarget mammalian cell lines with Bombyx mori macula-like virus.

Bombyx mori-derived cell lines are generally used for Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculovirus expression vector system (BEVS). However, almost all of the B. mori-derived cell lines are persistently infected with Bombyx mori macula-like virus (BmMLV). In this study, nontarget mammalian cell lines were exposed to BmMLV, and their susceptibility was investigated. Real-time PCR showed that viral RNA in virus-inoculated nine mammalian cell lines decreased sharply at 7 days postinfection. Also, there was no significant effect on cell viability of mammalian cells after inoculation with BmMLV. These findings indicate that mammalian cell lines used in this study are not permissive to BmMLV, and BmMLV contamination might not affect the safety aspect of BmNPV-based BEVS.

Identification of two juvenile hormone inducible transcription factors from the silkworm, Bombyx mori.

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mß (E(spl)mß), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mß was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mß was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mß are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.

Continuous cell lines from the common white, Pieris rapae crucivora Boisduval.

Three continuous cell lines, NIAS-PRC-819A, NIAS-PRC-819B, and NIAS-PRC-819C, were established from the pupal ovaries of the common white, Pieris rapae crucivora Boisduval (Insecta, Lepidoptera, Pieridae). The primary culture was initiated as explant cultures with ovariole fragments in MGM-464 medium supplemented with 20% fetal bovine serum at 25 degrees C. About 6 mo after the culture was set up, the first subculture was prepared. Thereafter, cells were subcultured with decreasing passage intervals, resulting in a cell population that multiplied continuously. The karyotypes of these cell lines were similar to each other, and the majority of the cells showed about 100 microchromosomes. The population-doubling times of these cell lines were 3 to 7 d. The cell lines were susceptible to a microsporidia, Nosema bombycis. Immunodiffusion experiments proved that these cell lines derived from the common white and not from other cell lines by contamination.

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua.

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.