The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Discovery and structure of a potent and highly specific blockerof insect calcium channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

Pest risk assessment of insects in sea cargo containers

A survey of the floors of 3001 empty sea cargo containers in storage was undertaken to estimate the quarantine risk of importing exotic insect pests into Australia, with special reference to pests of timber. More than 7400 live and dead insects were collected from 1174 containers. No live infestations of timber-feeding insects were recorded, but feeding damage detected in one floor indicates a low risk of importing colonies of timber pests in containers. The survey collection of dead insects demonstrates that containers are regularly exposed to economically important quarantinable insects, including timber pests (bostrichids, curculionids, cerambycids, siricids and termites), agricultural pests (including Adoretus sinicus, Adoretus sp., Carpophilus obsoletus and Philaenus spumarius), and nuisance pests (vespids and Solenopsis sp.). Stored product pests were found in more than 10% of containers. The assessment of pest risk associated with shipping containers is discussed in terms of the quantity and quality of opportunities for exotic insects to establish via this pathway.

Testing insect movement theory: empirical analysis of pest data routinely collected from agricultural crops

Observations of an insect's movement lead to theory on the insect's flight behaviour and the role of movement in the species' population dynamics. This theory leads to predictions of the way the population changes in time under different conditions. If a hypothesis on movement predicts a specific change in the population, then the hypothesis can be tested against observations of population change. Routine pest monitoring of agricultural crops provides a convenient source of data for studying movement into a region and among fields within a region. Examples of the use of statistical and computational methods for testing hypotheses with such data are presented. The types of questions that can be addressed with these methods and the limitations of pest monitoring data when used for this purpose are discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

Hydrolytic kinetic resolution of terminal mono- and bis-epoxides in the synthesis of insect pheromones: routes to (-)-(R)- and (+)-(S)-10-methyldodecyl acetate, (-)-(R)-10-methyl-2-tridecanone, (-)-(R)-(Z)-undec-6-en-2-ol (Nostrenol), (-)-(1R,7R)-1,7-dime

Hydrolytic kinetic resolution (HKR) of functionalised epoxides using (salen)Co(OAc) complexes provides enantiomerically enriched epoxides and diols, which have been transformed into important insect sex pheromones. In this general approach, (-)-(R)- and (+)-(S)-10-methyldodecyl acetates from the smaller tea tortrix moth were obtained, as was (-)-(R)-10-methyltridecan-2-one from the southern corn rootworm. The (S)-epoxide obtained from undec-1-en-6-yne was transformed to (-)-(R)-(Z)-undec-6-en-2-ol (Nostrenol) from ant-lions. HKR of appropriate bisepoxides was also investigated, and transformations of the resulting bisepoxides and epoxydiols provided (-)-(1R,7R)-1,7-dimethylnonylpropanoate from corn rootworms, (-)-(6R,12R)-6,12-dimethylpentadecan-2-one from the female banded cucumber beetle, and (-)-(2S,11S)-2,11-diacetoxytridecane and (+)-(2S,12S)-2,12-diacetoxytridecane from female pea-midges. (C) 2002 Elsevier Science Ltd. All rights reserved.

Simulation of insect movement with respect to plant architecture and morphogenesis

Developments in computer and three dimensional (3D) digitiser technologies have made it possible to keep track of the broad range of data required to simulate an insect moving around or over the highly heterogeneous habitat of a plant's surface. Properties of plant parts vary within a complex canopy architecture, and insect damage can induce further changes that affect an animal's movements, development and likelihood of survival. Models of plant architectural development based on Lindenmayer systems (L-systems) serve as dynamic platforms for simulation of insect movement, providing ail explicit model of the developing 3D structure of a plant as well as allowing physiological processes associated with plant growth and responses to damage to be described and Simulated. Simple examples of the use of the L-system formalism to model insect movement, operating Lit different spatial scales-from insects foraging on an individual plant to insects flying around plants in a field-are presented. Such models can be used to explore questions about the consequences of changes in environmental architecture and configuration on host finding, exploitation and its population consequences. In effect this model is a 'virtual ecosystem' laboratory to address local as well as landscape-level questions pertinent to plant-insect interactions, taking plant architecture into account. (C) 2002 Elsevier Science B.V. All rights reserved.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Application of quantitative analytical electron microscopy to the mineral content of insect cuticle

Quantification of calcium in the cuticle of the fly larva Exeretonevra angustifrons was undertaken at the micron scale using wavelength dispersive X-ray microanalysis, analytical standards, and a full matrix correction. Calcium and phosphorus were found to be present in the exoskeleton in a ratio that indicates amorphous calcium phosphate. This was confirmed through electron diffraction of the calcium-containing tissue. Due to the pragmatic difficulties of measuring light elements, it is not uncommon in the field of entomology to neglect the use of matrix corrections when performing microanalysis of bulk insect specimens. To determine, firstly, whether such a strategy affects the outcome and secondly, which matrix correction is preferable, phi-rho (z) and ZAF matrix corrections were contrasted with each other and without matrix correction. The best estimate of the mineral phase was found to be given by using the phi-rho (z) correction. When no correction was made, the ratio of Ca to P fell outside the range for amorphous calcium phosphate, possibly leading to flawed interpretation of the mineral form when used on its own.

Insect chemistry and chirality

Examination of the chemistry of a number of Australian insect species provided examples of unusual structures and encouraged determinations of their absolute stereochemistry by stereocontrolled syntheses and chromatographic comparisons. Inter alia, studies with the fruit-spotting bug (Amblypelta nitida), certain parasitic wasps (Biosteres sp.), the aposematic shield bug (Cantao parentum), and various species of scarab grubs are summarized. The determination of enantiomeric excesses (ee's) for component epoxides, lactones, spiroacetals, and allenes are described. Stereochemical and related aspects of the biosynthesis of spiroacetals in certain fruit-fly species (Bactrocerae sp.) are also presented.