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Development of recombinant viral insecticides by expression of an insect-specific toxin and insect-specific enzyme in nuclear polyhedrosis viruses.

As supplements to classical chemical insecticides, two approaches to develop recombinant baculovirus insecticides are described. In one approach an insect-specific toxin is expressed leading to a dramatic reduction in time to death. In the second approach an insect juvenile hormone esterase is expressed which leads to a reduction in feeding. Modifications of the wildtype esterase led to viruses which reduced the time to death as effectively as did the toxin-expressing virus. In both cases existing recombinant viruses are viewed as leads, and approaches to further improvement in the engineered viruses are suggested. Many of these approaches are based on analogy with the development of classical synthetic insecticides. Using these viruses as examples, the potential utility and limitations of recombinant viruses and other biological insecticides are discussed.

Amino Acid Sequence↗

Analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses.

We synthesized the cDNAs of an insect picornavirus, infectious flacherie virus of silkworm (IFV), genomic RNA and inserted it into a bacterial plasmid (pUC119). The 9,650 nucleotides (nts) sequence except for the poly(A) tail was obtained from the cloned cDNAs, and the sequence integrity was confirmed by primer extension and direct RNA sequencing. The sequence has a large open reading frame (ORF) of 9,255 nts (3,085 codons) flanked by the short 5' non-coding region (156 nts) and by the rather long 3' non-coding (239 nts). The structural proteins VP3, 4, 1 and 2 were located at the N-terminus of the polyprotein in this order and were preceded by a tentative small peptide. Computer analysis identified the sequences similar to the consensus sequences of 2C (helicase?), 3C (protease), and 3D (RNA polymerase) conserved among mammalian and plant picorna(-like) viruses. In addition, the predicted genome organization of IFV was quite similar to those of picornaviruses. Further analyses of the characteristics of the genome structure and a tentative phylogenetic tree constructed on the basis of the amino acid sequence similarity emphasized the evolutionary relationships among the insect and plant viruses.

Adenoviridae↗

Immunological studies of the effect of whole body insect extracts in the treatment of stinging insect allergy.

Specific IgE antibodies and total antibodies reacting with bee venom and yellow jacket venom were measured in sequential serum samples of insect-sensitive individuals. Venom-specific IgE decreased as a function of time and was not significantly affected by treatment with whole body extracts. There was no stimulation of total antibodies reacting with bee venom phospholipase A2 (PLA) following treatment with whole bee body extracts. These studies suggest that as measured by these parameters, whole body insect extracts used in the usual recommended doses are immunologically ineffective antigens.

Antibody Specificity↗

The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions.

To characterize the proteins involved in cell clump/cell adhesion of insect cellular defense reactions, we induced the cell clump/cell adhesion reaction in vitro with the hemolymph of larvae of the coleopteran insect, Tenebrio molitor. The 72 kDa protein was specifically enriched in the residues of cell clump/cell adhesion and was purified to homogeneity. A cDNA clone for the 72 kDa protein was isolated. We found that the 72 kDa protein was an activated phenoloxidase from Tenebrio pro-phenoloxidase. We suggest that activated phenoloxidase is involved in the cell clump/cell adhesion reaction as well as in the synthesis of melanin.

Amino Acid Sequence↗

Chemical-warfare techniques for insect control: insect 'pests' in Germany before and after World War I.

During World War I, chemical-warfare practices were introduced into economic entomology in Germany. Fritz Haber, 'the father of chemical warfare', realized that Germany could not win the war and thus looked for 'civilian' uses for his chemical arsenal. Before the war, there was a rhetoric of dangerous 'masses' of insects but the large-scale techniques needed to deal with them had not been developed. The gap between rhetoric and practices enabled entomology to integrate chemical weapons into its working methods. This article traces transformations in the ways of seeing insects and their control from the mid-nineteenth century to after World War I.

Animals↗

Insect repellent/antifeedant activity of 2,4-methanoproline and derivatives against a leaf- and seed-feeding pest insect.

2,4-methanoproline is a natural product isolated from the seeds of Ateleia herbert smithii Pittier that was formerly suggested to have insect repellent/antifeedant activity; however, this was not tested quantitatively. In this study the insect repellent/antifeedant potency of methanoproline was measured against larvae of the cotton leafworm, Spodoptera littoralis (Boisd.), and adults of the cowpea weevil, Callosobruchus maculatus (F.). In addition, several N-alkyl, amino, and nitrile derivatives of methanoproline with varying stereodemanding substituents were synthesized and also tested. It was shown that in S. littoralis methanoproline itself did not show any significant activity but that derivatives 5, 7, 8, and 10 did show a reasonable repulsive/antifeedant activity that was comparable to the commercial repellent DEET. A significant repellent activity was scored for methanoproline in adults of C. maculatus that was similar to DEET.

Animals↗

Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core alpha1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme.

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.

Allergens↗

Suitability of commercially available insect traps and pheromones for monitoring dusky sap beetles (Coleoptera: Nitidulidae) and related insects in Bt sweet corn.

Two trap types and pheromone sources for the dusky sap beetle, Carpophilus lugubris Murray (Coleoptera: Nitidulidae), were compared in Bacillus thuringiensis (Bt) and non-Bt sweet corn fields over a 3-yr period. Overall, commercial traps and pheromones were equally effective as experimental traps and pheromones used previously for capturing C. lugubris and other sap beetle species. The commercial trap often caught significantly more Glischrochilus quadrisignatus Say than the experimental trap that had been used in previous studies. Bt corn significantly reduced caterpillar damage to ears compared with the non-Bt isoline and did not adversely affect levels of Orius sp., the most common insect predator. Sap beetle damage was the most common insect damage to Bt sweet corn ears. Sap beetles were detected by traps at population levels below that which are likely to cause economic concern, indicating commercially available traps and pheromone lures for monitoring sap beetles should be suitable for detecting them under commercial growing conditions.

Bacillus thuringiensis Toxins↗

Efficacy of insect parasitoid Dirhinus himalayanus (Hymenoptera: Chalcididae) & insect growth regulator, triflumuron against house fly, Musca domestica (Diptera: Muscidae).

BACKGROUND & OBJECTIVES: In fly management programme chemicals are extensively used. Combined use of insect growth regulator (IGR) and parasitoids would yield a better reduction in fly density, as exposure of fly larvae to sub lethal dose of IGR enhances pupal duration and thereby increases the exposure time for parasitism. The objective of the present study was to explore the additive effect of both Dirhinus himalayanus, an insect parasitoid and triflumuron, an IGR in controlling house fly, Musca domestica. METHODS: In the field trial the study sites were allocated based on two-way ANOVA performed on one year pre-treatment survey. Parasitoid release and IGR treatment were initiated when the growth rate (lambda) of M. domestica was in an increasing trend. Parasitoids were released at a rate of 6 number/m2 for three months (February-April 2000), whereas IGR was applied at a dose of 10 mg/m2 at fortnightly interval from February to December 2000. Control efficacy was assessed using Mulla's formula. RESULTS: Reduction in puparia density was 59.29, 48.67 and 69.08 per cent respectively in areas, where parasitoids, ICR and combination of both IGR and parasitoids were used. Corresponding figures for adult population were 55.69, 49.71 and 77.14 per cent respectively. The per cent reduction in puparia and adult density was significantly (P<0.05) higher in the experimental areas than in the check. When the per cent reduction in adult and puparia density among different experimental areas was compared, it was observed that the reduction in fly density was significantly higher in the areas, where the combination of both parasitoid-IGR (P=0.00102) and IGR (P=0.03175) were used, while in areas where parasitoid (P=0.06191) were released the reduction in fly density was not significant. Similarly, there was a significant reduction in puparia density in areas where both parasitoid-IGR combination (P=0.0001) or IGR (P=0.002) were used, whereas in parasitoid (P=0.0612) released areas it was not significant. INTERPRETATION & CONCLUSION: The results show that the combined use of parasitoid and IGR is effective in reducing puparia and fly density. Therefore, for sustenance of an effective fly control programme, both parasitoid and IGR may be used.

Animals↗

[Insect bite allergy. Acute therapy and hyposensitization by means of insect venom].

Systemic reactions after an insect bite necessitate an allergologic investigation. In addition to taking the patient's history a skin test and an estimation of allergenspecific IgE should be performed. If insect bite allergy is confirmed immediate pharmacological treatment is necessary and hyposensitization therapy is recommended. Purified venom extracts are available.

Adrenal Cortex Hormones↗

Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.

The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.

Animals↗

Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines.

We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40. Unique eight base cutter restriction sites flanking the promoters and poly-adenylation sequences make it possible to transfer the entire transcription units into other sequence contexts, for example, into transposable elements or into other plasmids bearing selectable marker genes. It is also convenient to combine two of our transcription units on the same plasmid in order to express multiple genes simultaneously. To test the ability of our vectors to drive expression of reporter genes, luciferase derivatives were made of the expression plasmids and introduced into Aedes albopictus C6/36 cells by electroporation or into Anopheles gambiae embryos by biolistic particle bombardment. All three promoters directed high levels of luciferase expression. However, there were differences in their relative activities in the two experimental systems. In C6/36 cells, the actin 5C and hr5-ie1 promoters were significantly more active than the hsp70 promoter. In Anopheles embryos, hsp70 and actin 5C had maximal activities, while hr5-ie1 was weaker. We also found that the constructs containing the SV40 small t intron and early 3' untranslated region sequences had higher expression levels than their counterparts containing the Heliothis poly-adenylation sequence. Our most active construct combines the actin 5C promoter with the SV40 intron and 3' untranslated region sequences. This vector was also used to drive expression of a visible marker, the enhanced green fluorescent protein gene, resulting in readily visible green fluorescent protein expression in C6/36 cells.

3' Untranslated Regions↗

Intrinsic glycosylation potentials of insect cell cultures and insect larvae.

The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. The Spodoptera frugiperda (Sf21) and Trichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, the T. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus in T. ni larvae was modified with complex oligosaccharide having sialic acid linked alpha(2-6) to galactose.

Alkaline Phosphatase↗

Insect radiosensitivity: dose curves and dose-fractionation studies of dominant lethal mutations in the mature sperm of 4 insect species.

Males of 4 species of insects: Musca domestica L. (housefly) (Diptera), Oncopeltus fasciatus (Dallas) ( milkweed bug) (Hemiptera), Anagasta kuhniella ( Zeller ) ( mealmouth ) (Lepidoptera) and Heliothis virescens (Fab.) (tobacco budworm ) (Lepidoptera) were irradiated as adults. Dose--response curves for the induction of dominant lethal mutations in the mature sperm were constructed. The curves were analyzed mathematically and compared with theoretical computer simulated curves requiring 1, 2, 4, 8 and 16 'hits' for the induction of a dominant lethal mutation. The 4 species belonging to 3 different orders of insects showed a wide range in radiation sensitivity and vastly different dose--response curves. The house fly was the most sensitive and displayed a '1-hit' curve. The milkweed bug was intermediate in sensitivity and the curve exhibited '4-hit' kinetics. The mealmoth and tobacco budworm were most radioresistant and the response curves resembled 8-16--hit simulated curves. When the data were analyzed by several mathematical models we found that a logistic response curve gave reasonably good fit with vastly different parameters for the 4 species. Dose-fractionation experiments showed no reduction in the frequency of lethal mutations induced in any species when an acute dose was fractionated into 2 equal exposures separated by an 8-h period. Various reasons for large differences in radiosensitivity are discussed, e.g., (1) differences in repair mechanisms, (2) variation in oxygen concentration, (3) holokinetic versus monokinetic chromosomes and (4) intrinsic differences in mitotic cell cycles which affect the behavior of radiation-induced chromosome breaks.

Animals↗

Actions of potent cholinergic anthelmintics (morantel, pyrantel and levamisole) on an identified insect neurone reveal pharmacological differences between nematode and insect acetylcholine receptors.

Intracellular recording and current-clamp techniques were used to investigate the cholinergic activity of the anthelmintics, morantel, pyrantel and levamisole, applied to the fast coxal depressor motorneurone (Df) of the cockroach Periplaneta americana. Application of these agents and acetylcholine to the bath resulted in dose-dependent changes in conductance and corresponding depolarization of the neuronal membrane. Relative potencies of the drugs were determined from dose-response relationships and the rank order of effectiveness was as follows: carbachol much greater than levamisole greater than pyrantel greater than morantel. Evidence that these drugs were acting at the same site of action was obtained with the antagonist, mecamylamine, which abolished the responses to all these agents. It is concluded that the weak insecticidal action of these potent anthelmintics may result in part from their weak cholinergic agonist action on insect neurones, which contrasts with their potent agonist actions on acetylcholine receptors of helminth nerve and muscle tissues. The striking differences in potency on different invertebrate tissues appears to reflect differences in the properties of acetylcholine receptors between insects and nematodes. Further characterization of neurotransmitter receptors in invertebrates is needed in order to facilitate the rational design of broad-spectrum antiparasitic agents with low toxicity in mammals.

Animals↗

Insect acetyl-CoA carboxylase: activity during the larval, pupal and adult stages of insect development.

1. The activity of the lipogenic enzyme, acetyl-CoA carboxylase, was investigated in four insect species; Bombyx mori (Lepidoptera), Tenebrio molitor (Coleoptera), Glossina morsitans and Sarcophaga nodosa (Diptera). 2. Acetyl-CoA carboxylase activity in larval, pupal and adult forms was compared with the saponifiable lipid mass at each stage of the life-cycle, and found to follow similar patterns except for Tenebrio molitor. 3. The results are examined in relation to known metabolic requirements for each insect.

Acetyl-CoA Carboxylase↗

Plant-insect interactions: molecular approaches to insect resistance.

Recent advances in our understanding of induced responses in plants and their regulation, brought about by a revolution in molecular biology, have re-focused attention on the potential exploitation of endogenous resistance mechanisms for crop protection. The future goal of crop biotechnology is thus to engineer a durable, multimechanistic resistance to insect pests through an understanding of the diversity of plant responses to insect attack.

Animals↗

Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion.

The mannose-specific snowdrop lectin (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph. The ability of GNA to act as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1: SFI1) to the haemolymph of lepidopteran larvae was investigated. Constructs encoding SFI1 and an SFI1/GNA fusion protein were expressed in Pichia pastoris. The insecticidal activity of purified recombinant proteins on injection was found to be comparable to published values for SfI1 purified from spider venom [Toxicon 40 (2002) 125]. Whereas neither GNA nor SFI1 alone showed acute toxicity when fed to larvae of tomato moth (Lacanobia oleracea), feeding SFI1/GNA fusion at 2.5% of dietary proteins was insecticidal to first stadium larvae, causing 100% mortality after 6 days. The protein also showed a significant, dose dependent, toxicity towards fourth and fifth stadium larvae, with growth reduced by up to approximately 90% over a 4-day assay period compared to controls. Delivery of intact SFI1/GNA to the haemolymph in these insects was shown by western blotting; haemolymph samples from fusion-fed larvae contained a GNA-immunoreactive protein of the same molecular weight as the SFI1/GNA fusion. SFI1/GNA and similar fusion proteins offer a novel and effective approach for delivering haemolymph active toxins by oral administration, which could be used in crop protection by expression in transgenic plants.

Animals↗