Dipteran Cell Lines Research Articles

Quantitative evaluation of the molting hormone activity in coleopteran cells established from the Colorado potato beetle, Leptinotarsa decemlineata

A novel reporter gene assay system using BCIRL-Lepd-SL1, a cell line from the coleopteran Colorado potato beetle (Leptinotarsa decemlineata), was established. Cells were transiently transfected with the reporter plasmid that was composed of a firefly luciferase gene with upstream ecdysone response elements, and an internal control plasmid that constitutively produces Renilla reniformis luciferase. Transfected cells were incubated with various molting hormone agonists, and the activity of these agonists was quantitatively determined by measuring luminescence emission. Transcription-inducing activity for ecdysone, 20-hydroxyecdysone and ponasterone A in terms of EC50 (50% effective concentration) were determined to be 1μM (pEC50=5.99), 68nM (pEC50=7.17) and 1.3nM (pEC50=8.88), respectively. Among tested diacylhydrazine (DAH)-type compounds, 11 compounds were active (pEC50=3.56∼6.41), but two compounds were inactive. The EC50 values were linearly correlated to their binding affinity except for one compound. While several ecdysone reporter systems were developed before that employ dipteran and lepidopteron cell lines, the assay system described here is only the second one that employs a coleopteran cell line. This reporter system will allow screening in high-throughput format for new and more potent molting accelerating compounds that specifically target coleopteran pests.

Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells

Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1-5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.

Examining the relative activity of several dicistrovirus intergenic internal ribosome entry site elements in uninfected insect and mammalian cell lines

Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 5' IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.

Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

ACMNPV IN PERMISSIVE, SEMIPERMISSIVE, AND NONPERMISSIVE CELL LINES FROM ARTHROPODA

Insect cell lines from Arthropoda represented by Lepidoptera, Coleoptera, Diptera, and Homoptera were evaluated for their ability to support replication of AcMNPV. In addition, some of the cell lines that were refractive to AcMNPV were tested with AcMNPV hsp70 Red, a recombinant carrying the red fluorescent protein (RFP) gene, for their ability to express this protein after inoculation. Of the 10 lepidopteran cell lines tested, only three cell lines from Helicoverpa zea (BCIRL-HZ-AM1), Lymantria dispar (IPLB-LD 65), and Cydia pomonella (CP-169) failed to support detectable viral replication as measured by tissue culture infectious dose 50 (TCID50) assay. Heliothis virescens (BCIRL-HV-AM1) produced the highest viral titer of 2.3 +/- 0.1 x 10(7) TCID50/ml followed by Heliothis subflexa (BCIRL-HS-AM1) at 4.7 +/- 0.1 x 10(6) TCID50/ml and Spodoptera frugiperda (IPLB-SF21) at 4.1 +/- 0.1 x 10(6) TCID50/ml. None of the coleopteran, dipteran, or homopteran cell lines supported AcMNPV replication. However, when studies were performed using AcMNPV hsp70 Red, the dipteran cell lines Aedes aegypti (ATC-10) and Drosophila melanogaster (line 2), both expressed the RFP as well as the refractive lepidopteran cell lines from H. zea and L. dispar. No RFP expression was observed in any of the coleopteran or homopteran cell lines. Cell lines refractive to AcMNPV did not appear to be adversely affected by the virus, as judged by their ability to multiply, nor was there any indication of induced apoptosis, as assessed by deoxyribonucleic acid fragmentation profiles or cell blebbing, or both.

Selective mechanism of action of tebufenozide on lepidopteran cell lines

The non-steroidal ecdysone agonist, tebufenozide (RH-5992), induces a precocious incomplete molt primarily on lepidopteran insects, but has little or no effect on insects of other orders. 20 Hydroxyecdysone at 10−7 M induced the transcription factor CHR3 mRNA in CF-203 cells and DHR3 mRNA in DM-2 cells. Tebufenozide even at 10−10 M induced CHR3 mRNA in lepidopteran CF-203 cells, but even at 10−5 M it induced only trace levels of DHR3 mRNA in dipteran DM-2 cells. Studies using radiolabelled RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than dipteran cell lines (DM-2 and Kc). The efflux of radiolabelled RH-5992 from DM-2 cells was temperature-dependent and was blocked by 10−5 M ouabain, an inhibitor of Na+, K+-ATPase, suggesting that the efflux was due to active transport.

Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and K c, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10 −7 M 20E induced CHR3 mRNA in CF-203 cells and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10 −10 M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10 −6 M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C- RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and K c). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10 −5 M ouabain, an inhibitor of Na +, K +-ATPase suggesting that the efflux was due to active transport.

A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines

A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines. Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus ( OpMNPV) immediate-early 2 ( ie2) promoter. Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines. A chimeric synthetic bacterial- OpMNPV ie promoter-Zeocin ™ resistance gene cassette has been included to facilitate cloning in E. coli as well as the generation of stably transformed insect cell lines. The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Baculovirus immediate-early promoter-mediated expression of the Zeocin ™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 μg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 μg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus ( OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus ( AcMNPV) were used to drive expression of the Zeocin resistance gene ( ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3–4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by β-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.

Characterization of clonal populations of theHeliothis zea cell line IPLB-HZ 1075

A total of 24 clones (HZ 1075/UND-A through X) were initially isolated by dilution plating from the established IPLB-HZ 1075 cell line. Many of the isolates with highly vacuolated cytoplasms eventually died during subculturing. The cloned cell strains differed in their predominant morphology, cell doubling times, and relative ability to support replication of the singly encapsulated nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). The origin of the cloned cell strains was confirmed by comparing their isozyme profiles with those of the parental IPLB-HZ 1075 cell line andH. zea larvae using stains for fumerate hydratase, lactate dehydrogenase (LDH), and malic dehydrogenase (MDH). One dipteran and several lepidopteran cell lines maintained in our lab were also separable using stains for LDH and MDH.

Factors to consider in performing survival studies with insect cells.

Insect cell lines are not well-suited to colony formation in liquid medium following low-density cell plating. The present studies demonstrate that the time of addition of fetal bovine serum to the culture medium and the number of gamma-irradiated feeder cells added to each plate are important factors in developing a useful colony formation assay. TN-368 lepidopteran and WR69-DM-1 dipteran cell lines were used for these experiments. Both cell types display increased plating efficiencies if serum is added to the medium one or more days prior to plating as compared to adding serum immediately before plating. Growth curves obtained by seeding cells at higher densities also indicate that cell growth is slightly better if serum is added one or more days before seeding. These findings are especially important for survival and toxicity studies because the results demonstrate that even seemingly minor factors involved in cell survival assays may benefit treated cells to a greater degree than untreated control cells, thus providing an erroneous assessment of cell survival.

Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin.

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

Radiosensitivity of Cultured Insect Cells: II. Diptera

The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

A coleopteran cell line derived fromHeteronychus arator (Coleoptera: Scarabaeidae)

The first continuous coleopteran cell line, designated DSIR-HA-1179, was derived from the scarab beetleHeteronychus arator. Most cells are spindle-shaped, 25 to 40 µm long and 15 to 20 µm wide, adhere strongly to plastic surfaces, and are highly resistant to trypsin and collagenase treatment. The population doubling time of 6 d, at 27° C, is slow compared with most dipteran and lepidopteran cell lines. The cells cross-react in comparative radioimmunoassays withH. arator larval tissue but not other insect cell lines and support the replication ofOryctes baculovirus and at least two nodaviruses, black beetle virus, and Flock House virus.

Characterization of invertebrate cell lines

The usefulness of isozyme patterns for distinguishing 14 lepidopteran and 2 dipteran cell lines was evaluated. The lepidopteran cell lines used in this study represent eight taxonomic families with one family, Noctuidae, having five representatives. Cell extracts were examined for 18 isozymes using a starch gel electrophoretic system. Ten isozymes proved to be suitable because their isozyme patterns permitted the allocation of the cell lines into distinct groups. Furthermore, four isozymes (isocitrate dehydrogenase, malic enzyme, phosphoglucoisomerase, and phosphoglucomutase) were found to be adequate to distinguish the cell lines. The isozyme patterns observed for the two dipteran and one of the lepidopteran cell lines were analogous to the profiles found using the intact insect. Isozyme analyses differentiated the cell lines and may prove useful for identifications of species of origin. The use of this technique as a criterion for identification of invertebrate cell lines is proposed.