Establishment and characterization of insect cell lines from 10 lepidopteran species

Summary Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera, the velvetbean caterpillar,Anticarsia gemmatalis, the corn earworm,Helicoverpa zea, the tobacco budworm,Heliothis virescens, the beet armyworm,Spodoptera exigua, the fall armyworm,Spodoptera frugiperda, and the cabbage looper,Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines fromA. ipsilon andH. virescens being adapted to suspension culture, using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion sex, age, marital status, or handicap.     

In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Summary The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AMI and TN-CLI cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.     

The underestimated N-glycomes of lepidopteran species

BackgroundInsects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability.MethodsUsing an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line.ResultsWe detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed.ConclusionThe lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species.SignificanceThe occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.     

Isolation and identification of 20-hydroxyecdysone from a lepidopteran continuous cell line

Extracts of three continuous cell lines from the cabbage looper, Trichoplusia ni, were assayed for the presence of ecdysteroids. While no evidence of ecdysteroids was present in the extracts of the ovarian (TN-368) or embryonic (IPLB-TN-R²) cell lines, radioimmunoassays on extracts of media and extracts of cell pellets from imaginal disc cell cultures (IAL-TND1) were positive. The immunoreactive material from both cells and media co-migrated with a 20-hydroxyecdysone standard on reversed-phase high-performance liquid chromatography (HPLC). The immunoreactive fractions from the cell extract were chromatographed on silica HPLC and subjected to mass spectral analysis. Both of these analyses indicated that the unknown compound was 20-hydroxyecdysone. Radioimmunoassay indicated up to 28 ng of ecdysone equivalents in cells (3.75 x 10⁷ cells) from 50 ml of IAL-TND1 cultures, which is equivalent to 120 ng of 20-hydroxyecdysone based on relative reactivity of the antiserum used in this study. This report presents the first evidence of 20-hydroxyecdysone production by a continuous insect cell line and also the first to show that cells from imaginal discs are capable of ecdysteroid synthesis.     

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly,Papilio polyxenes

The black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro-oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro-oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH-dependent glutathione peroxidases (selenium-dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium-independent glutathione-S-transferase [GT_px]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX-like activity which reduces both H₂O₂ and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H₂O₂, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.     

Activities of enzymes that detoxify superoxide anion and related toxic oxyradicals in Trichoplusia ni

In third-, fourth-, and fifth-instar larvae of the cabbage looper moth, Trichoplusia ni, the activities of the antioxidant enzymes, superoxide dismutase (SOD*), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) were examined using 850 g supernatants of whole-body homogenates. The enzyme activities, expressed as units mg⁻¹ protein min⁻¹ at 25°C ranged as follows: SOD, 0.67-2.13 units; CAT, 180.5-307.5 units; GPOX, none detectable; and GR, 0.40-1.19 units. There was a similar pattern of changes for SOD and CAT activities with larval ontogeny, but not for GR. The cabbage looper apparently uses SOD and CAT to form a “defensive team” effective against endogenously produced superoxide anion (O₂⪸). Glutathione may serve as an antioxidant for the destruction of any organic/lipid peroxides formed, and GSH oxidized to glutathione disulfide would be recycled by GR. Bioassays against pro-oxidant compounds exogenous sources of (O₂⪸) show high sensitivity of mid-fifth instars to the linear furanocoumarin, 8-methoxypsoralen (xanthotoxin) primarily from photoactivation (320-380 nm), and auto-oxidation of the flavonoid, quercetin. The LC₅₀s are 0.0004 and 0.0045% (w/w) concentration of xanthotoxin and quercetin, respectively. Both pro-oxidants have multiple target sites for lethal action and, in this context, the role of antioxidant enzymes is discussed.     

Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, produced in cells grown in vitro

A virus isolated from the alfalfa looper, Autographa californica, replicated successfully and rapidly in a suspended ovarian cell line of the cabbage looper, Trichoplusia ni. Polyhedra were observed in the nucleus of cells within 20 hr after inoculation. The cytopathological changes typical of nuclear polyhedrosis infections were observed, and an average of 64 polyhedra/cell were produced. These polyhedra were quantitatively as infectious to cabbage looper larvae as those produced in vivo. In addition, they were infective to Heliothis virescens, Pectinophora gossypiella, Spodoptera exigua, A. californica, and Anagrapha falcifera.     

A high‐quality chromosome‐level genome assembly of a generalist herbivore,Trichoplusia ni

The cabbage looper, Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest. T. ni has evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticide Bacillus thuringiensis (Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333‐Mb high‐quality T. ni genome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein‐coding genes. High‐density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis of T. ni with Bombyx mori showed enrichment of tandemly duplicated genes in T. ni in families involved in detoxification and digestion, consistent with the broad host range of T. ni. High levels of genome synteny were found between T. ni and other sequenced lepidopterans. However, genome synteny analysis of T. ni and the T. ni derived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high‐quality T. ni genome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect‐plant interactions and co‐evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.     

Specificity of cultured insect tissue cells for bioassay of entomocidal protein fromBacillus thuringiensis

Summary Cultured tissue cells from lepidopteran and dipteran sources displayed an order-specific response to entomocidal protein from crystals ofBacillus thuringiensis. Protein isolated from crystals ofB. thuringiensis subsp.kurstaki was effective against cells of the spruce budworm (Choristoneura fumiferana) and the tobacco hornworm (Manduca sexta), but was inactive against both mosquito cell lines tested (Aedes aegypti andAnopheles gambiae). Conversely, protein from inclusion bodies ofB. thuringiensis subsp.israelensis was fully active only against the mosquito cell lines but displayed reduced (four- to seven-fold) toxicity for the lepidopteran cell lines. One exception to this pattern of specificity was observed with aPlodia interpunctella cell line, which failed to respond to either crystal protein preparation. The moth toxin was stable at 4° C for months, whereas the mosquito toxin was susceptible to proteolytic degradation and was unstable for periods longer than 2 wk.     

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA

Summary Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R², IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.     

Characterization of a new continuous cell line from the flood water mosquito,Aedes vexans

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (=2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

The establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae)

Summary The history and characteristics of two cell lines developed from primary explants of pupal tissue from the insect,Spodoptera frugiperda (J. E. Smith), are described. One cell line, IPLB-SF-21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolymph-free medium at the 6th passage. The IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common to tissue antigens from pupae ofSpodoptera frugiperda. At the time this work was done, Ronald H. Goodwin was a Postdoctoral Research Fellow sponsored by the National Academy of Sciences. Mention of a proprietary product does not constitute endorsement by the U.S. Department of Agriculture.     

Establishment of a cell line derived from embryos of the potato tuber mothPhthorimaea operculella (Zeller)

Summary A cell line from the main insect pest of potatoes in tropical and subtropical areas,Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace’s modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of theAutographa californica nuclear polyhedrosis virus.     

Production of selected baculoviruses in newly established lepidopteran cell lines

Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×10⁶TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×10⁶TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

Chitinolytic enzymes from Streptomyces albidoflavus expressed in tomato plants: effects on Trichoplusia ni

Tomato (Lycopersicon esculentum ) cultivars were transformed with genes that encode bacterial chitinolytic enzymes (i.e., endochitinase and chitobiosidase) from Streptomyces albidoflavus. Transgenic tomato plants producing these enzymes were found to have enhanced resistance to cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), consistently reducing the growth rates of larvae. Mortality was significantly increased in two of three feeding trials. Ingestion of endochitinase and chitobiosidase not only affected development of larval T. ni from neonate to ultimate instar, but they also caused mortality and decreased insect weight when exposure began during the third instar. The results of this study provide some insight into the mode of action of the chitinolytic enzymes, by supporting the hypothesis that ingested chitinolytic enzymes damage the chitin component of the peritrophic envelope, leading to increased permeability. The size of marker molecules (FITC-dextrans) that permeated the peritrophic envelopes of T. ni feeding on transgenic plants were 50% larger than those permeating the peritrophic envelopes of T. ni feeding on the control plants. Further research is needed to more clearly identify the sites and modes of action of these chitinolytic enzymes, and the potential for synergy between these enzymes and pathogens, allelochemicals, and other environmental factors.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Highly passage of <Emphasis Type="Italic">Spodoptera litura</Emphasis> 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.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.