Representative honey bee viruses do not replicate in the small hive beetle, Aethina tumida Murray.

The small hive beetle (SHB), Aethina tumida Murray, is an invasive pest of the honey bee and causes significant damage through the consumption of colony resources and brood. Two assumptions related to honey bee virus transmission have been made about SHB: first, that SHB vectors honey bee viruses and second, that these viruses replicate in SHB based on the detection of both positive and negative strand viral genomic RNA within SHB. To clarify the role of SHB in virus transmission, we sought to address whether selected honey bee viruses replicate in SHB. Sequences derived from five honey bee viruses were identified in the transcriptomes of field-caught SHB from the U.S., but not in those of lab-reared SHB, suggesting that these viruses do not replicate in SHB. To elucidate whether the representative viruses, Israeli acute paralysis virus (IAPV; Dicistroviridae) and Deformed wing virus (DWV; Iflaviridae) replicate in SHB, we tested for replication in vitro in an SHB-derived cell line (BCIRL-AtumEN-1129-D6). Following treatment of the cell line with viral particles or viral RNA, the number of virus genomes was monitored by reverse transcription quantitative PCR (RT-qPCR). In contrast to the positive control, IAPV and DWV RNA levels steadily decreased over a period of 8 days. Collectively, these results from bioinformatic observations and in vitro experiments indicate that IAPV and DWV do not replicate in SHB. These results are consistent with the host specificity of most insect viruses within a single insect order and indicate that while SHB may serve as a mechanical vector of honey bee viruses within and between hives, this insect does not serve as a biological vector for these honey bee viruses.

A cell line derived from the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae).

Insect cell lines are effective tools used in industry and academia. For example, they are used in screening potential insecticides, in making certain proteins for biomedical applications, and in basic research into insect biology. So far, there are no cell lines derived from the black soldier fly, Hermetia illucens (BSF). This may become an issue because BSFs are employed in a range of industrial and household processes. BSFs are used in producing biodiesel, in developing cosmetics and skin creams, and in the production of some medicines and animal feeds. BSF larvae process waste streams from a variety of sources into food for some animals and are also used in household composting. Our BSF cell line, designated BCIRL-HiE0122021-SGS, was developed from eggs using the medium CLG#2 (50% L-15 + 50% EX-CELL 420, with 9% FBS and antibiotics), with many other media being tested. This cell line consists of attached cells with a variety of morphologies and its identity was authenticated using CO1 barcoding. A growth curve was generated and the resulting doubling time was 118 h. We quantified the fatty acid methyl esters (FAMES) and recorded the expected range of saturated, monounsaturated, and polyunsaturated FAMEs, with only trace levels of lauric acid being noted. The BSF cell line is available free of charge by request.

Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

φC31 -Mediated cassette exchange in Sf9 insect cells for stable expression.

Insect cells, especially Sf9 cells, are commonly used in biomanufacturing due to their advantages in high expression levels and post-translational modification. However, the development of stable expression cell lines via random integration tended to be unstable. Site-specific integration (SSI) is an alternative strategy. In this study, a φC31 -mediated cassette exchange system in Sf9 cells was established for SSI. The tagging cassette with the reporter gene egfp was randomly inserted into the cell genome. Potential platform cell lines were obtained by fluorescence-activated cell sorting (FACS) and single-cell cloning. Platform cell lines were selected by assessing the fluorescence expression, stability, and growth kinetics of cell lines. The selected platform cell lines were co-transfected with the φC31-containing plasmid and the targeting cassette. Green-fluorescence-negative clones were screened by hygromycin resistance and FACS. The resulting cell clones exhibited the expression properties of the platform cell lines. The rapid development of cell lines for the production of influenza subunit vaccines by the cassette exchange system demonstrated that the system constituted a versatile and reusable platform for the production of various recombinant proteins. Overall, the φC31-mediated cassette exchange system in Sf9 cells has the potential to facilitate and accelerate biologics development.

Prostaglandin A2 induces apoptosis in three cell lines derived from the fall armyworm, Spodoptera frugiperda.

Animals maintain homeostasis of cell numbers, constantly creating new cells and eliminating others. Programmed cell death, apoptosis, is a mechanism of cell elimination and it acts in many aspects of animal biology. Drawing on the biomedical background, several signals launch the apoptosis mechanisms, including prostaglandins (PGs). Based on this information, we posed the hypothesis that PGs similarly induce apoptosis in insect cell lines. We used three Spodoptera frugiperda cell lines, including two newly established, BCIRL-SfNS-0518B-YL derived from the central nervous system and BCIRL-Sf4FB-0614-SGS derived from fat body, and the commercially available Sf9 cells. Using a kinetic apoptosis kit, we found treating SfNS cells for 18 h with 15 or 20 µM PGA2 led to decreases in cell numbers, coupled with increased numbers of apoptotic and dead cells. Similar exposures to 10 µM PGA2 (24 h) led to substantial increases in apoptotic cells, confirmed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay on a flow cytometer. The influence of PGA2 treatments increased with dosage, as we recorded about 20% apoptosis at 24 h post-PGA2 treatments (10 µM) and about 34% apoptosis at 24 h post-30 µM treatments. PGA2 treatments led to 10- to 30-fold increases in messenger RNAs (mRNAs) encoding apoptosis-specific caspases-1, -2, -3, and -5 at 12 h and 40- to 60-fold increases in mRNAs encoding caspases-1 and -2, 10-fold increases for caspases-3 and -5 at 24 h. These findings strongly support our hypothesis that PGs induce apoptosis in an insect cell line and confirm an additional PG action in insect biology.

Screening of single-cell clonal lines from Papilio demoleus Linnaeus cell lines for exogenous protein expression and adaptation in serum-free culture.

In this paper, four established cell lines derived from newly hatched larvae of Papilio demoleus Linnaeus and 57 single-cell clones derived from the 3 lines were used as test materials. Recombinant ß-galactosidase baculovirus AcMNPV-Gal was used to infect the P. demoleus L. cell lines and the single-cell clones, and recombinant protein expression in each cell line was detected and compared. Three clonal cell lines, RIRI-PaDe-1-C1, RIRI-PaDe-2-C6 and RIRI-PaDe-3-C52, which showed significantly higher ß-galactosidase expression levels than those of the parental cell lines, were selected. Five types of commercial serum-free media for insect cells, Express Five SFM, Ex-Cell 405, Sf-900III SFM, Sf-900II SFM and HyClone Serum-Free Media, were used to adapt RIRI-PaDe-2-C6 cells and RIRI-PaDe-3-C52 cells to serum-free culture conditions, and the growth characteristics of the cells and the exogenous protein expression characteristics before and after adaptation were compared. The results showed that RIRI-PaDe-2-C6 cells could stably proliferate in Ex-Cell 405, RIRI-PaDe-3-C52 cells could stably proliferate in Express Five SFM and Ex-Cell 405, and the rate of proliferation of and the level of expression of ß-galactosidase in RIRI-PaDe-3-C52 cells were significantly increased in Express Five SFM.

Prostaglandins influence protein phosphorylation in established insect cell line.

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid and two other C20 polyunsaturated fatty acids. Among other actions in invertebrates, PGs act in ovarian development, renal functions, immunity, hemocyte migration, and gene/protein expression. Reversible phosphorylation is a major mechanism of regulating protein functions in eukaryotic cells and for some mammalian proteins it is influenced by PGs. We posed the hypothesis that PGs influence protein phosphorylation within insect cells, which we tested with the established insect cell line, BCIRL-HzAM1. After 20, 30, or 40 min incubations in the presence of one of three PGs (at 15 µM), PGA2 , PGE1 , or PGF2α , separate sets of cells were processed for analysis by two-dimensional electrophoresis followed by tandem mass spectrometry. We recorded significant phosphorylation changes in 31 proteins, decreases in 15, and increases in 15, and one protein with increased or decreased phosphorylation, depending on PG treatment. Increasing PG exposure times led to changes in fewer proteins, 20 min incubations led to changes in 16 proteins, 30 min to changes in 13, and 40 min to changes in 2 proteins. The proteins were identified by bioinformatic analyses, including transcript description, calculated molecular weights and isoelectric points, MOlecular Weight SEarch score, total ion score, numbers of peptides, percent protein coverage, E-value, and highest peptide score. The data presented in this paper firmly support our hypothesis that PGs influence protein phosphorylation within insect cells and adds a novel PG-signaled function to insect biology.

Identification, molecular characterization and expression of aminopeptidase N-1 (APN-1) from Anopheles stephensi in SF9 cell line as a candidate molecule for developing a vaccine that interrupt malaria transmission.

BACKGROUND: According to the World Health Organization reports, billions of people around the world are at risk for malaria disease and it is important to consider the preventive strategies for protecting the people that are living in high risk areas. One of the main reasons of disease survival is diversity of vectors and parasites in different malaria regions that have their specific features, behaviour and biology. Therefore, specific regional strategies are necessary for successful control of malaria. One of the tools that needs to be developed for elimination and prevention of reintroduction of malaria is a vaccine that interrupt malaria transmission (VIMTs). VIMT is a broad concept that should be adjusted to the biological characteristics of the disease in each region. One type of VIMT is a vector-based vaccine that affects the sexual stage of Plasmodium life cycle. According to recent studies, the aminopeptidase N-1 of Anopheles gambiae (AgAPN-1) is as a potent vector-based VIMT with considerable inhibition activity against the sexual stage of Plasmodium parasite. METHODS: Systems for rapid amplification of cDNA ends (3'-RACE) and genome walking methods were used for sequence determination of apn-1 gene from Anopheles stephensi and distinct bioinformatics software were used for structural analysis. AsAPN-1 was expressed in Spodoptera frugiperda (Sf9) insect cell line using the baculovirus expression system. Recombinant AsAPN-1 was purified under the hybrid condition and its biological activity was assayed. RESULTS: Asapn-1 gene and its coded protein from An. stephensi were characterized for the first time in this study. Subsequently, the structural features and immunological properties of its coded protein were evaluated by in silico approaches. Enzymatic activity of the recombinant AsAPN-1, which was expressed in Sf9 insect cell line, was equal to 6 unit/µl. CONCLUSIONS: Results of this study revealed that AsAPN-1 is very similar to its counterpart in An. gambiae. In silico evaluation and fundamental data which are necessary for its evaluation as a VIMT-based vaccine in the next steps were acquired in this study and those could be useful for research groups that study on malaria vaccine for countries that An. stephensi is the main malaria vector there.

GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility.

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells.

Effects of lipopolysaccharide and juvenile hormone III treatments on cell growth and gene expression in the Ceratitis capitata (Diptera: Tephritidae) CCE/CC128 cell line.

The Mediterranean fruit fly Ceratitis capitata is one of the most important insect pest species in the world. It has a high colonization capacity and population variety, giving it considerable genetic diversity. Strategies for its control have included the sterile insect technique and insect growth regulators. Many studies have analyzed the medfly transcriptome, and along with the medfly genome sequence, the sequences of multiple genes related to sex determination, mating, development, pheromone detection, immunity, or stress have been identified. In this study, the medfly CCE/CC128 cell line was used to assess cell growth variation and changes in the expression of genes covering different functions, after lipopolysaccharide (LPS) and juvenile hormone III (JHIII) treatments. No significant effects on cell growth and gene expression were observed in the cells treated with LPS. In the cells treated with JHIII, the results showed significant effects on cell growth, and an overexpression was found of the Shade gene, one of the Halloween gene members of the cytochrome p450 family, which is involved in development and the synthesis of 20-hydroxyecdysone. This study shows preliminary results on the insect cell line in combination with whole-genome sequencing, which can facilitate studies regarding growth, toxicity, immunity, and transcriptome regulations as a response to different compounds and environmental alterations.

Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda (Sf), is a polyphagous lepidopteran herbivore that consumes more than 80 plant species, including many economically important crops, such as corn, soybeans, and sorghum. While already a serious pest in the Americas, it was recently introduced into Africa, India, and China. Because of its high economic costs in the New World and the continent-wide damage potentials in Africa, research to develop advanced pest management technologies is necessary. We are supporting this need by developing novel, next-generation insect cell lines from targeted tissues. Cell lines, such as these, will boost insecticide discovery programs and lead to innovative pest management solutions. Here, we report on the establishment of 16 new cell lines from larval S. frugiperda tissues: nine from the central nervous system, three from the aorta, and four from the testes. We confirmed the identities of the cell lines by DNA amplification fingerprinting polymerase chain reaction, determined their doubling times from growth curves, and described cell types via microscopy. We also developed 16 sublines from three neuronal cell lines.

Transcriptional response of immune-related genes after endogenous expression of VP1 and exogenous exposure to VP1-based VLPs and CPV virions in lepidopteran cell lines.

In insects, RNAi is considered the major antiviral immune defense pathway. DsRNAs produced during viral infection are processed by Dicer enzymes into small RNAs that function as specificity determinants to silence viral genes. By contrast, in mammals, recognition of molecules associated with viral infection, such as dsRNA, by pattern recognition receptors (PRRs) initiates a signaling cascade that culminates in the production and release of signaling proteins with antiviral function such as interferons. However, in insects, the hypothesis that components of virions can be recognized as pathogen-activated molecular patterns (PAMPs) to activate the innate immune response has not been investigated systematically. In this study, the potential of VP1, that constitutes the major capsid protein of cytoplasmic polyhedrosis virus (CPV; Reoviridae), to activate a collection of immune-related genes was examined in silkworm-derived Bm5 cells. Two different methods of VP1 administration were tested, either through endogenous expression in transformed cell lines, or through addition of purified VP1-based viral-like particles to the extracellular medium. In addition, exposure to CPV virions isolated from purified polyhedra was also performed. In general, our results do not show a robust transcriptional response of immune-related genes to VP1 or CPV virions, but two exceptions were noted. First, the expression of the antimicrobial peptide (AMP) gene Attacin was strongly induced after 24 h of exposure to VP1-based VLPs. Second, the expression levels of dcr-2, an essential gene in the RNAi pathway, were greatly increased in VP1-expressing transformed Sf21 cells but not transformed Bm5 cells, indicating the existence of species-specific effects. However, the increased expression of dcr-2 did not result in increased silencing efficiency when tested in an RNAi reporter assay. Our study indicates that the capsid protein VP1 of CPV has the potential to act as a PAMP and to induce a transcriptional response in insect cells that relate both to RNAi and protein effectors such as AMPs. The identity of the PRRs and the signaling cascade that are potentially triggered by VP1 remain to be elucidated in future experiments. While this study was performed on a small scale, it can encourage more comprehensive studies with high-throughput approaches (microarray, deep sequencing) to search more systematically whether viral capsid proteins can act as PAMPs in insects and whether their production results in the induction of immune-related genes with potential antiviral function.

The full-length Cry1Ac protoxin without proteolytic activation exhibits toxicity against insect cell line CF-203.

The new dual model for Bacillus thuringiensis insecticidal mechanism proposed that Cry1A protoxins without proteolytic activation could bind to insect midgut receptors to exert toxicity. To evaluate insecticidal potency of Cry1Ac protoxin at precluding interference of midgut proteases, the cytotoxicity of Cry1Ac protoxin against midgut cell line CF-203 derived from Choristoneura fumiferana was analyzed. It was revealed that Cry1Ac protoxin was toxic to CF-203 cells and there existed certain differences in the cytological changes when treated with protoxin and toxin. Our cell-based study provided direct evidence for the proposed dual model and shed light on exploring the difference between two toxic pathways elicited by intact protoxin and activated toxin.

Establishment of an embryonic cell line from the American cockroach Periplaneta americana (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process.

Despite the pest status and medicinal value of the American cockroach Periplaneta americana, few attempts have been made to establish cell lines from this insect owing to the difficulty of culturing Blattarian cells. Here, we describe the establishment of the RIRI-PA1 line from P. americana embryo tissue following primary culture in modified Grace's medium containing 20% fetal bovine serum. RIRI-PA1 was found to primarily consist of attached spindle-shaped and giant cells, which attach themselves to their container. The population-doubling time of 40th-passage cells was approximately 84.8 h. The average chromosome number at the 30th passage was 42, with 40% of cells demonstrating substantial variations, with the highest number of variations of 78 and lowest of 24. The identity of RIRI-PA1 was confirmed by comparing the COI gene of these cells to that of P. americana embryo tissue. Telomerase activity decreased in primary cells after 7 d of culture and 5th-passage cells in comparison to embryo tissues; however, compared to the other cultured cells tested, the telomerase activity significantly increased at the 20th passage. We propose that the stagnation periods and cessation of proliferation observed relate to cellular telomerase activity, but the relationship between insect cell proliferation and telomerase as well as the regulatory mechanism involved remains to be elucidated.

Expression of three reporter genes in four cell lines developed from Papilio demoleus Linnaeus (Lepidoptera: Papilionidae).

This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), ß-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells. The inoculation densities of the tested cell lines were 2 × 104 cells/well (96-well plate) and 1 × 105 cells/well (24-well plate), and adding a volume of virus stock resulted in an MOI of 5.0. The results showed that the four cell lines could be infected by recombinant baculovirus and that cell lysis occurred 96 h after infection. In the four tested cell lines, only a small number of RIRI-PaDe-1 and RIRI-PaDe-3 cells expressed recombinant GFP and showed green fluorescence. The expression was much lower than that of Sf9 and High Five. Comparing the intracellular and extracellular activity of ß-galactosidase indicated that the P. demoleus cell system was more suitable for the expression of secreted proteins, and its extracellular ß-galactosidase level was close to that of Sf9, but the expression level of SEAP was far lower than those of Sf9 and High Five.

RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles.

Conformationally complex membrane proteins (MPs) are therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of virus-like particles (VLPs), preserving their native lipidic environment. Here, we implemented a site-specific recombinase-mediated cassette exchange (RMCE) strategy to establish a reusable HIV-1 Gag-expressing insect cell line for fast production of target MPs on the surface of Gag-VLPs. The Sf9 cell line was initially tagged with a Gag-GFP-expressing cassette incorporating two flipase recognition target sites (FRTs), one within the fusion linker of Gag-GFP. The GFP cassette was afterwards replaced by a Cherry cassette via flipase (Flp) recombination. The fusion of Gag to fluorescent proteins enabled high-throughput screening of cells with higher Gag expression and Flp-mediated cassette exchange ability, while keeping the functionality of the VLP scaffold unaltered. The best cell clone was then Flp-recombinated to produce Gag-VLPs decorated with a human ß2-adrenergic receptor (ß2AR). Release of a fluorescently labeled ß2AR into the culture supernatant was confirmed by immunoblotting, and its co-localization with Gag-VLPs was visualized by confocal microscopy. Furthermore, the differential avidity of ß2AR-dsplaying Gag-VLPs versus "naked" Gag-VLPs to an anti-ß2AR antibody measured by ELISA corroborated the presence of ß2AR at the surface of the Gag-VLPs. In conclusion, this novel insect cell line represents a valuable platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.

Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 105 cells ml-1) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 107 TCID50 ml-1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera).

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 µm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

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.