Pyridalyl, a novel insecticide: potency and insecticidal selectivity

Pyridalyl is an insecticide of a novel chemical class (unclassified insecticides). Toxicity of pyridalyl to two insect pest species, Spodoptera litura and Frankliniella occidentalis, an insect predator, Orius stringicollis, and a pollinator, Bombus terrestris, was evaluated in the laboratory. The insecticidal activity of pyridalyl against S. litura was evaluated using the leaf-dipping method. The potency of pyridalyl was highly effective against all development stages (2nd to 6th instar larvae) of S. litura. This compound was also evaluated against F. occidentalis using the direct spray method. For F. occidentalis, toxicity of pyridalyl was almost similar to that of acrinathrin, but greater than acrinathrin for adults. Then the toxicity of this product to the natural enemies, Orius stringicollis and the pollinating insect Bombus terrestris, was evaluated using the body-dipping method or direct spray method. No acute toxicity of this product was observed on these non-target insects. Moreover, the influence of pyridalyl to the nest of Bombus terrestris was evaluated using the direct spray to the inside of the nest. No apparent influence of this compound was observed by 21 days after treatment. The cytotoxicity of pyridalyl to the Sf9 insect cell line and the CHO-K1 mammalian cell line was evaluated using the trypan-blue exclusion method. High toxicity to the insect cell line, but almost no toxicity to the mammalian cell line, was observed. Thus, pyridalyl exhibited high selectivity in cytotoxicity between the insect and mammalian cell line as well as in insecticidal activity among insect species. We infer pyridalyl may be useful for IPM programs of greenhouse cultivation system.     

Selective action of acetogenin mitochondrial complex I inhibitors

Five annonaceous acetogenins, rolliniastatin-1 [structure: see text], rolliniastatin-2 [structure: see text], laherradurin [structure: see text], squamocin [structure: see text], annonacin [structure: see text], and rotenone as a reference, differing in their NADH oxidase inhibition activity, have been evaluated for antifeedant, insecticidal, trypanocidal and cytotoxic effects on insect, mammalian and tumor cells. All the test compounds were toxic to Leptinotarsa decemlineata, demonstrated selective cytotoxicity to insect Sf9 cells and a panel of tumor cell lines with the multidrug-resistant SW480 (P-glycoprotein+, Pgp+) being the most sensitive one. Compounds [structure: see text] and rotenone had post-ingestive effects against Spodoptera littoralis larvae while [structure: see text] and rotenone were active against Trypanosoma cruzi. Based on their biochemical properties (inhibition of the mitochondrial NADH oxidase activity), the in vivo effects of these compounds on S. littoralis and their cytotoxic effects on Sf9 and tumor cells were more predictable than their effect on T. cruzi and mammalian cells.     

In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

The monoclonal antibody (mAb) CO17‐1A specifically binds to the tumor‐associated cell surface glycoprotein GA733 in colorectal cancer cells. Thus, mAb CO17‐1A has the potential to act as an immune therapeutic protein against colorectal cancer. Recently, it was shown that the baculovirus insect cell expression system produces anti‐colorectal cancer mAb CO17‐1A. In this study, the colorectal cancer antibody mAb CO17‐1A fused to the endoplasmic reticulum (ER) retention signal sequence (KDEL), and the (mAb CO17‐1AK) was expressed in Spodoptera frugiperda Sf9 insect cells. The yield, cell cytotoxicity, and in vitro anti‐tumor activity of mAb CO17‐1AK were verified. Western blotting was performed to confirm that both heavy and light chains of mAb CO17‐1A were expressed in Sf9 insect cells. The insect‐derived mAb (mAb^I) CO17‐1A was purified using a protein G affinity column. An in vitro wound healing assay was conducted to determine the inhibition activity of mAb CO17‐1A during tumor cell migration, showing that mAb^I CO17‐1AK was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration.     

Expression and purification of biologically active v-sis/platelet-derived growth factor B protein by using a baculovirus vector system.

Malignant transformation induced by simian sarcoma virus is mediated by its v-sis protein, the monkey homolog of the platelet-derived growth factor (PDGF) B chain. By use of an appropriately engineered baculovirus expression vector, the v-sis protein was expressed in the insect cell line Spodoptera frugiperda (Sf9) at a level 50- to 100-fold higher than that observed with overexpression in mammalian-cell transfectants. The sis protein produced by Sf9 cells underwent processing similar to that observed in mammalian cells, including efficient disulfide-linked dimer formation. Moreover, the recombinant sis protein was capable of binding PDGF receptors and inducing DNA synthesis as efficiently as PDGF-B synthesized by mammalian cells. A significant fraction of sis protein was released from Sf9 cells, which made possible a one-step immunoaffinity purification to near homogeneity with a 40% recovery of biological activity. These results demonstrate that a protein whose normal processing requires both intrachain and interchain disulfide-bridge formation can be efficiently expressed in a biologically active form in insect cells by using a baculovirus vector system.     

Toxicity,membrane binding and uptake of the <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> agglutinin (SSA) in different insect cell lines

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC₅₀) ranging between 9 and 42 μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.     

Baculovirus expression system: An alternative for producing catalytically active human PTP-1B

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.     

The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin.

The Bacillus thuringiensis CryIAa toxin binds a cadherin-like protein (BtR175) on the brush-border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin-binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.     

Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.     

Benzylideneacetone and other phenylethylamide bacterial metabolites induce apoptosis to kill insects

Xenorhabdus and Photorhabdus are entomopathogenic bacteria that can induce immunosuppression against target insects by suppressing eicosanoid biosynthesis, leading to fatal septicemia. These bacteria can synthesize and release secondary metabolites such as benzylideneacetone (BZA) and other phenylethylamide compounds that can inhibit phospholipase A₂ (PLA₂) and shut down eicosanoid biosynthesis. However, insecticidal activities of these bacterial metabolites remain unclear. Thus, the objective of this study was to assess cytotoxicities of BZA and seven other bacterial metabolites to insect cells. These eight bacterial metabolites exhibited significant cytotoxicities against an insect cell line Sf9 at micromolar range. Especially, BZA and cPY were highly potent at low micromolar range. When these eight bacterial metabolites were injected to hemocoels of Spodoptera exigua larvae, they significantly decreased total count of hemocytes. In Sf9 cell line and hemocytes, these bacterial metabolites induced cell membrane blebbings, apoptotic vesicles, and genomic DNA fragmentation. Terminal deoxyribonucleotidyl transferase nick end translation assay showed that these bacterial metabolites caused significant DNA breakages in cells in a dose-dependent manner. However, a pan caspase inhibitor treatment significantly rescued the cell death induced by these bacterial metabolites. Cytotoxicities of these bacterial metabolites were highly correlated with their insecticidal activities. These results indicate that the insecticidal activities of the bacterial metabolites may be induced by their apoptotic activities against hemocytes and other insect cells. Taken together, these results suggest that phenylethylamide compounds might have potential as novel insecticides.     

Existence of catalase-less peroxisomes in Sf21 insect cells

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.     

Biological Characterization of a Nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) Isolated from the Philippines

A Philippine Spodoptera litura nucleopolyhedrovirus (SltMNPV) was isolated by plaque purification from an uncloned NPV population in a formulated product that exhibits high insecticidal activity against the onion-attacking common cutworm, S. litura. Its estimated genome size was approximately 142 kb. Comparison with Autographa californica MNPV and Spodoptera exigua NPV by restriction fragmentation analysis showed that it was a distinct isolate. Light microscopy examination showed that SltMNPV-P7 infected insect cell lines derived from Spodoptera littoralis (CLS-79), Spodoptera frugiperda (Sf9), and S. litura (TUAT-SpLi221). It produced typical cytopathic effects in cell lines established from Bombyx mori and S. exigua and induced apoptotic-like cytopathology in Lymantria dispar-derived cell line; it exerted no observable cytopathic effect on Spilosoma imparilis cell line. Significant increase in budded virus (BV) production was observed in CLS-79, Sf9, and TUAT-SpLi221 cell lines, in which TUAT-SpLi221 cell line supported the highest BV titer. No significant BV production was observed in the remaining four cell lines. Similarly, viral DNA replication and polyhedrin production proceeded only in these three spodopteran cell lines. Results showed that SltMNPV-P7 is a unique isolate which can be propagated in vitro for further studies and thus has a potential for development into a more effective microbial insecticide.     

The molecular action of the novel insecticide, Pyridalyl

Pyridalyl is a recently discovered insecticide that exhibits high insecticidal activity against Lepidoptera and Thysanoptera. Pyridalyl action requires cytochrome P450 activity, possibly for production of a bioactive derivative, Pyridalyl metabolism being prevented by general P450 inhibitors. Apoptosis is apparently not involved in the cytotoxicity. Continuous culture of Spodoptera frugiperda Sf21 cells in sub-lethal doses of Pyridalyl, results in a Pyridalyl-resistant cell line. Probing the molecular action of Pyridalyl by comparison of the proteomes of Pyridalyl-resistant and -susceptible cell lines, revealed differential expression of a number of proteins, including the up-regulation of thiol peroxiredoxin (TPx), in the resistant cells. Treatment of Bombyx mori larvae with Pyridalyl, followed by comparison of the midgut microsomal sub-proteome, revealed the up-regulation of three proteasome subunits. Such subunits, together with Hsp70 stress proteins, glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) and thiol peroxiredoxin (TPx) were also up-regulated in the whole proteome of B. mori BM36 cells following treatment with the insecticide. The foregoing results lead to the hypothesis that cytochrome P450 action leads to an active Pyridalyl metabolite, which results in production of reactive oxygen species (ROS), that leads to damage to cellular macromolecules (e.g., proteins) and enhanced proteasome activity leads to increased protein degradation and necrotic cell death.     

The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.     

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.     

Triterpene-based plant defenses

Pentacyclic triterpenes are abundant in the plant kingdom and have a wide array of pharmacological activities. They also have insect antifeedant effects and therefore apparently play a role in plant defense. In this paper, we describe the insecticidal activity of pentacyclic triterpenes of plant origin from different chemical classes on several insect pests (Spodoptera littoralis, Leptinotarsa decemlineata and Myzus persicae), their phytotoxic properties and their selective cytotoxic effects on insect-derived Sf9 and mammalian CHO cells. We also discuss the role they play in plant defense based on these activities.     

Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II

Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation.     

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein

Summary The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinantAutographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established fromSpodoptera frugiperda, Trichoplusia ni, andMamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in theM. brassicae-derived MB0503 cell line was Endo H resistant. The commonly usedS. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.