A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

The biometeorology of high-altitude insect layers.

Flight at high altitude is part of a migration strategy that maximises insect population displacement. This thesis represents the first substantial analysis of insect migration and layering in Europe. Vertical-looking entomological radar has revealed specific characteristics of high-altitude flight: in particular layering (where a large proportion of the migrating insects are concentrated in a narrow altitude band). The meteorological mechanisms underpinning the formation of these layers are the focus of this thesis. Aerial netting samples and radar data revealed four distinct periods of high-altitude insect migration: dawn, daytime, dusk, and night-time. The most frequently observed nocturnal profiles during the summertime were layers. It is hypothesised that nocturnal layers initiate at a critical altitude (200–500 m above ground level) and time (20:00–22:00 hours UTC). Case study analysis, statistical analysis, and a Lagrangian trajectory model showed that nocturnal insect layers probably result from the insects’ response to meteorological conditions. Temperature was the variable most correlated with nocturnal insect layer presence and intensity because insects are poikilothermic, and temperatures experienced during high-altitude migration in temperate climates are expected to be marginal for many insects’ flight. Hierarchical effects were detected such that other variables—specifically wind speed—were only correlated with insect layer presence and intensity once temperatures were warm. The trajectory model developed comprised: (i) insect flight characteristics; (ii) turbulent winds (which cause vertical spread of the layer); and (iii) mean wind speed, which normally leads to horizontal displacements of hundreds of kilometres in a single migratory flight. This thesis has revealed that there is considerable migratory activity over the UK in the summer months, and a range of fascinating phenomena can be observed (including layers). The UK has moved from one of the least studied to perhaps the best studied environments of aerial insect migration and layering in the world.

Layers of nocturnal insect migrants at high-altitude: the influence of atmospheric conditions on their formation

1 Radar studies of nocturnal insect migration have often found that the migrants tend to form well-defined horizontal layers at a particular altitude. 2 In previous short-term studies, nocturnal layers were usually observed to occur at the same altitude as certain meteorological features, most notably at the altitudes of temperature inversions or nocturnal wind jets. 3 Statistical analyses are presented of four years’ data that compared the presence, sharpness and duration of nocturnal layer profiles (observed using continuously-operating entomological radar) with meteorological variables at typical layer altitudes over the UK. 4 Analysis of these large datasets demonstrated that temperature was the foremost meteorological factor persistently associated with the presence and formation of longer-lasting and sharper layers of migrating insects over southern UK.

Tracking larval insect movement within soil using high resolution X-ray microtomography

1. In contrast to above-ground insects, comparatively little is known about the behaviour of subterranean insects, due largely to the difficulty of studying them in situ. 2. The movement of newly hatched (neonate) clover root weevil (Sitona lepidus L. Coleoptera: Curculinidae) larvae was studied non-invasively using recently developed high resolution X-ray microtomography. 3. The movement and final position of S. lepidus larvae in the soil was reliably established using X-ray microtomography, when compared with larval positions that were determined by destructively sectioning the soil column. 4. Newly hatched S. lepidus larvae were seen to attack the root rhizobial nodules of their host plant, white clover (Trifolium repens L.). Sitona lepidus larvae travelled between 9 and 27 mm in 9 h at a mean speed of 1.8 mm h(-1). 5. Sitona lepidus larvae did not move through the soil in a linear manner, but changed trajectory in both the lateral and vertical planes.

Genetically modified insect resistance in cotton: some farm level economic impacts in India

The paper explores the impact of insect-resistant Bacillus thuringiensis (Bt) cotton on costs and returns over the first two seasons of its commercial release in three sub-regions of Maharashtra State, India. It is the first such research conducted in India based on farmers' own practices rather than trial plots. Data were collected for a total of 7793 cotton plots in 2002 and 1577 plots in 2003. Results suggest that while the cost of cotton seed was much higher for farmers growing Bt cotton relative to those growing non-Bt cotton, the costs of bollworm spray were much lower. While Bt plots had greater costs (seed plus insecticide) than non-Bt plots, the yields and revenue from Bt plots were much higher than those of non-Bt plots (some 39% and 63% higher in 2002 and 2003, respectively). Overall, the gross margins of Bt plots were some 43% (2002) and 73% (2003) higher than those of non-Bt plots, although there was some variation between the three sub-regions of the state. The results suggest that Bt cotton has provided substantial benefits for farmers in India over the 2 years, but there are questions as to whether these benefits are sustainable. (c) 2004 Elsevier Ltd. All rights reserved.

Dynamic scaling of bred vectors in spatially extended chaotic systems

We unfold a profound relationship between the dynamics of finite-size perturbations in spatially extended chaotic systems and the universality class of Kardar-Parisi-Zhang (KPZ). We show how this relationship can be exploited to obtain a complete theoretical description of the bred vectors dynamics. The existence of characteristic length/time scales, the spatial extent of spatial correlations and how to time it, and the role of the breeding amplitude are all analyzed in the light of our theory. Implications to weather forecasting based on ensembles of initial conditions are also discussed.

Moist singular vectors and the predictability of some high impact European cyclones

The ECMWF full-physics and dry singular vector (SV) packages, using a dry energy norm and a 1-day optimization time, are applied to four high impact European cyclones of recent years that were almost universally badly forecast in the short range. It is shown that these full-physics SVs are much more relevant to severe cyclonic development than those based on dry dynamics plus boundary layer alone. The crucial extra ingredient is the representation of large-scale latent heat release. The severe winter storms all have a long, nearly straight region of high baroclinicity stretching across the Atlantic towards Europe, with a tongue of very high moisture content on its equatorward flank. In each case some of the final-time top SV structures pick out the region of the actual storm. The initial structures were generally located in the mid- to low troposphere. Forecasts based on initial conditions perturbed by moist SVs with opposite signs and various amplitudes show the range of possible 1-day outcomes for reasonable magnitudes of forecast error. In each case one of the perturbation structures gave a forecast very much closer to the actual storm than the control forecast. Deductions are made about the predictability of high-impact extratropical cyclone events. Implications are drawn for the short-range forecast problem and suggestions made for one practicable way to approach short-range ensemble forecasting. Copyright © 2005 Royal Meteorological Society.

The influence of physical processes on extratropical singular vectors

An investigation is made of the impact of a full linearized physical (moist) parameterization package on extratropical singular vectors (SVs) using the ECMWF integrated forecasting system (IFS). Comparison is made for one particular period with a dry physical package including only vertical diffusion and surface drag. The crucial extra ingredient in the full package is found to be the large-scale latent heat release. Consistent with basic theory, its inclusion results in a shift to smaller horizontal scales and enhanced growth for the SVs. Whereas, for the dry SVs, T42 resolution is sufficient, the moist SVs require T63 to resolve their structure and growth. A 24-h optimization time appears to be appropriate for the moist SVs because of the larger growth of moist SVs compared with dry SVs. Like dry SVs, moist SVs tend to occur in regions of high baroclinicity, but their location is also influenced by the availability of moisture. The most rapidly growing SVs appear to enhance or reduce large-scale rain in regions ahead of major cold fronts. The enhancement occurs in and ahead of a cyclonic perturbation and the reduction in and ahead of an anticyclonic perturbation. Most of the moist SVs for this situation are slightly modified versions of the dry SVs. However, some occur in new locations and have particularly confined structures. The most rapidly growing SV is shown to exhibit quite linear behavior in the nonlinear model as it grows from 0.5 to 12 hPa in 1 day. For 5 times this amplitude the structure is similar but the growth is about half as the perturbation damps a potential vorticity (PV) trough or produces a cutoff, depending on its sign.

Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands

Despite widespread concern about declines in pollination services, little is known about the patterns of change in most pollinator assemblages. By studying bee and hoverfly assemblages in Britain and the Netherlands, we found evidence of declines (pre- versus post-1980) in local bee diversity in both countries; however, divergent trends were observed in hoverflies. Depending on the assemblage and location, pollinator declines were most frequent in habitat and flower specialists, in univoltine species, and/or in nonmigrants. In conjunction with this evidence, outcrossing plant species that are reliant on the declining pollinators have themselves declined relative to other plant species. Taken together, these findings strongly suggest a causal connection between local extinctions of functionally linked plant and pollinator species.

Multitrophic links between arbuscular mycorrhizal fungi and insect parasitoids

The effects of arbuscular mycorrhizal colonization of Leucanthemum vulgare on parasitism of a leaf-mining insect was studied in a field and a laboratory experiment. In the field, parasitism of Chromatomyia syngenesiae by Diglyphus isaea was lower on mycorrhizal plants, compared with plants where the association was reduced. A laboratory experiment, in which L. vulgare was inoculated with three species of AM fungi, showed that the effects on parasitism rates were mycorrhizal species-dependent. Some fungal combinations increased parasitism, some decreased it, while others had no effect. It is concluded that the most likely cause of these differences is plant size, with parasitoid searching efficiency being reduced on the larger plants, resulting from certain mycorrhizal species combinations. However, a mycorrhizal effect on herbivore-produced plant volatiles cannot be ruled out.

Susceptibility of different insect pupae to the bacterial symbiont, Xenorhabdus nematophila, isolated from the entomopathogenic nematode Steinernema carpocapsae

Cells of the bacterial symbiont Xenorhabdus nematophila from the entomopathogenic nematode, Steinernema carpocapsae entered the pupae of Plutella xylostella after 15 minutes treatment with suspensions containing the bacterial cells. Secretions of Xenorhabdus nematophila, in either broth or water, were found lethal to the pupae of P. xylostella when applied in moist sand. The bacterial symbiont Xenorhabdus nematophila was found lethal to the pupae of greater wax moth (Galleria mellonella), beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella) and black vine weevil (Otiorhynchus sulcatus) in the absence of the nematode vector and the cells of X. nematophila entered the haemocoele of the pupae.

Genetically modified insect resistance in cotton: some farm level economic impacts in India

The paper explores the impact of insect-resistant Bacillus thuringiensis (Bt) cotton on costs and returns over the first two seasons of its commercial release in three sub-regions of Maharashtra State, India. It is the first such research conducted in India based on farmers' own practices rather than trial plots. Data were collected for a total of 7793 cotton plots in 2002 and 1577 plots in 2003. Results suggest that while the cost of cotton seed was much higher for farmers growing Bt cotton relative to those growing non-Bt cotton, the costs of bollworm spray were much lower. While Bt plots had greater costs (seed plus insecticide) than non-Bt plots, the yields and revenue from Bt plots were much higher than those of non-Bt plots (some 39% and 63% higher in 2002 and 2003, respectively). Overall, the gross margins of Bt plots were some 43% (2002) and 73% (2003) higher than those of non-Bt plots, although there was some variation between the three sub-regions of the state. The results suggest that Bt cotton has provided substantial benefits for farmers in India over the 2 years, but there are questions as to whether these benefits are sustainable. (c) 2004 Elsevier Ltd. All rights reserved.