Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Central projections of Drosophila sensory neurons in the transition from embryo to larva

Sensory axons of different sensory modalities project into typical domains within insect ganglia. Tactile and gustatory axons project into a ventral layer of neuropil and proprioceptive afferents, including chordotonal axone, into an intermediate or dorsal layer. Here, we describe the central projections of sensory neurons in the first instar Drosophila larva, relating them to the projection of the same sensory afferents in the embryo and to sensory afferents of similar type in other insects. Several neurons show marked morphologic changes in their axon terminals in the transition between the embryo and larva. During a short morphogenetic period late in embryogenesis, the axon terminals of the dorsal bipolar dendrite stretch receptor change their shape and their distribution within the neuromere. In the larva, external sense organ neurons (es) project their axons into a ventral layer of neuropil. Chordotonal sensory neurons (ch) project into a slightly more dorsal region that is comparable to their projection in adults. The multiple dendrite (md) neurons show two distinctive classes of projection. One group of md neurons projects into the ventral-most neuropil region, the same region into which es neurons project. Members of this group are related by lineage to es neurons or share a requirement for expression of the same proneural gene during development. Other md neurons project into a more dorsal region. Sensory receptors projecting into dorsal neuropil possibly provide proprioceptive feedback from the periphery to central motorneurons and are candidates for future genetic and cellular analysis of simple neural circuitry. J. Comp. Neurol. 425:34-44, 2000. (C) 2000 Wiley-Liss, Inc.

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Foraging and vein-cutting behaviour of Euploea core corinna (W.S. Macleay) (Lepidoptera : Nymphalidae) caterpillars feeding on latex-bearing leaves

Caterpillars of Euploea core corinna (W. S. Macleay) sever leaf veins prior to feeding on their latex-bearing host plants, which restricts the flow of latex at feeding sites. The severing of leaf veins by insects feeding on latex-bearing plants is commonly referred to as 'sabotaging' and is thought to be an evolved response by the insect to counter the negative effects of feeding on latex-rich leaves. Sabotaging behaviour is described for all instars of E. core corinna, with particular attention given to neonates. Vein cutting by neonate E. core corinna caterpillars can occur within 2 h of hatching, with most caterpillars establishing feeding sites within 10 h. Commonly, first instars cut an are-shaped row of leaf side-veins parallel to the leaf margin, but they may also cut the leaf mid-rib in a fashion similar to older instar larvae. From a sample of 50 E. core corinna larvae, representing all instars, we found that the diameters of the veins cut by caterpillars are closely correlated to larval head width (r=0.90). Through manipulative experiments, we demonstrate for the first time that sabotaging behaviour in neonate caterpillars imposes no detectable short-term physiological costs on those caterpillars.

Onnamide F: A new nematocide from a southern Australian marine sponge, Trachycladus laevispirulifer

A southern Australian marine sponge, Trachycladus laevispirulifer, has yielded a potent new nematocide with antifungal activity which has been identified as onnamide F (1). The structure for 1 was assigned by detailed spectroscopic analysis and chemical conversion to the methyl ester 2. Onnamide F contains a common structural motif previously described in a number of natural products exhibiting interesting pharmacological activities, including the insect chemical defense agent pederin (3), and the sponge metabolites the onnamides, mycalamides, and theopederins.

Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

Promoter studies of a polydnavirus gene from Cotesia rubecula (Hym : Braconidae)

The Cotesia rubecula polydnavirus gene, CrV1, is expressed in a highly transient fashion. Within four hours after egg deposition and virus infection, tissues of the host caterpillar, Pieris rapae, express high levels of the transcript. Twelve hours after infection no transcripts are visible. We have previously shown that the CrV1 secreted protein is mainly produced in host haemocytes. In haemocytes, immune functions such as phagocytosis and cell spreading are abolished by destabilization of the cell cytoskeleton. To test whether the observed down-regulation of CrV1 transcripts is mediated by transcriptional control or by other factors, such as the disruption of cytoskeleton in CrV1-inactivated cells, we cloned the promoter and the 3' untranslated region of the CrV1 gene to study CrV1 expression. The promoter region of the CrV1 gene was cloned into baculovirus expression systems along with the CAT reporter gene. Molecular analyses showed that the CAT gene under the control of CrV1 promoter is expressed as early as 2 h post infection and continues until late phase of infection suggesting that down-regulation of CrV1 expression in host haemocytes is perhaps mediated by post-transcriptional mechanisms.

Sequence analysis and expression of a virus-like particle protein,VLP2, from the parasitic wasp Venturia canescens

Endoparasitoid wasps produce maternal protein secretions, which are transported into the body of insect hosts at oviposition to regulate host physiology for successful development of their offspring. Venturia canescens calyx fluid contains so-called virus-like particles (VLPs) that are essential for immune evasion of the developing parasitoid inside the host. VLPs consist of four major proteins. In this paper, we describe the isolation and molecular cloning of a gene (vlp2) that is a constituent of VLPs and discuss its possible role in VLP structure and function.

Isolation and characterization of a neprilysin-like protein from Venturia canescens virus-like particles

Maternal protein secretions from endoparasitoid wasps are evolutionary adaptations to regulate host physiology as part of an extended wasp phenotype. Virus-like particles (VLPs) produced in the calyx region of Venturia canescens wasps are involved in immune evasion of the developing parasitoid inside the host. In contrast to polydnaviruses (PDVs), VcVLPs are devoid of any nucleic acids. To understand the role of these particles in the regulation of host physiology and phylogenetic relationship between VLPs and PDVs, it is essential to identify particle proteins. In this paper, we describe the isolation and molecular cloning of a neprilysin-like gene (VcNEP) coding for a 94 kDa VcVLP protein and discuss its possible role in host regulation.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the 131 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 mug/10(6) cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity. (C) 2002 Wiley Periodicals, Inc.

Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym : Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparositoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidose. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C rubecula PDV-protein (Crp32). Arch. Insect Biochem. Physiol. 53:92-100, 2003. (C) 2003 Wiley-Liss, Inc.

A comparison of the performance and biological efficiency of new knapsack sprayers and a controlled droplet application (CDA) sprayer for the control of the diamondback moth, Plutella xylostella L. (Lepidoptera : Plutellidae)

Field trials and laboratory bioassays were undertaken to compare the performance and efficacy (mortality of diamondback moth larvae) of insecticides applied to cabbages with three high volume hydraulic knapsack sprayers (NS-16, PB-20 and Selecta 12V) and a controlled droplet application (CDA) sprayer. In field experiments, the high volume knapsack sprayers (application rate 500-600 L ha(-1)) provided better spray coverage on the upper and lower surfaces of inner leaves, the upper surfaces of middle and outer leaves, and greater biological efficacy than the CDA sprayer (application rate 20similar to40 L ha(-1)). The PB-20 provided better spray coverage on the upper surface of middle leaves and both surfaces of outer leaves when compared with the Selecta 12V. However, its biological efficacy in the field was not significantly different from that of the other high volume sprayers. Increasing the application rate from 20 to 40 L ha(-1) for the CDA sprayer significantly increased droplet density but had no impact on test insect mortality. Laboratory evaluations of biological efficacy yielded higher estimates than field evaluations and there was no significant difference between the performance of the PB-20 and the CDA sprayer. Significant positive relationships were detected between insect mortality and droplet density deposited for both the PB-20 and the CDA sprayers.

The gall-inducing habit has evolved multiple times among the eriococcid scale insects (Sternorrhyncha : Coccoidea : Eriococcidae)

The habit of inducing plant galls has evolved multiple times among insects but most species diversity occurs in only a few groups, such as gall midges and gall wasps. This phylogenetic clustering may reflect adaptive radiations in insect groups in which the trait has evolved. Alternatively, multiple independent origins of galling may suggest a selective advantage to the habit. We use DNA sequence data to examine the origins of galling among the most speciose group of gall-inducing scale insects, the eriococcids. We determine that the galling habit has evolved multiple times, including four times in Australian taxa, suggesting that there has been a selective advantage to galling in Australia. Additionally, although most gall-inducing eriococcid species occur on Myrtaceae, we found that lineages feeding on Myrtaceae are no more likely to have evolved the galling habit than those feeding on other plant groups. However, most gall-inducing species-richness is clustered in only two clades (Apiomorpha and Lachnodius + Opisthoscelis), all of which occur exclusively on Eucalyptus s.s. The Eriococcidae and the large genus Eriococcus were determined to be non-monophyletic and each will require revision. (C) 2004 The Linnean Society of London.