Colin J. Alexander

The spatial dynamics of crop and ground active predatory arthropods and their aphid prey in winter wheat

The distribution of aphid predators within arable fields has been previously examined using pitfall traps. With this technique predominantly larger invertebrate species are captured, especially Carabidae, but the technique provides no estimate of density unless mark-recapture is used. However, many other numerically important aphid predators occur in arable fields and relatively little is known about their distribution patterns nor whether they exhibit a density-dependent response to patches of cereal aphids. Identification of the most effective predators can allow management practices to be developed accordingly. In this study, the distribution of cereal aphids and their predators was examined by suction sampling within a field of winter wheat in Devon, UK, along with visual estimates of weed patchiness. Sampling was conducted on four occasions in 1999 across a grid of 128 sample locations. The distribution of 11 predatory taxa from the Carabidae, Staphylinidae and Linyphiidae was examined. Additionally, the total number of aphid predators and a predation index were used in these analyses. Carabid adults and larvae, along with staphylinid larvae showed the strongest aggregation into patches and the most temporal stability in their distribution. Other taxa had more ephemeral distributions as did the cereal aphids. The distribution of carabid larvae was disassociated from the distribution of cereal aphids for the first two sampling occasions indicating biocontrol was occurring. Other predatory groups showed both association and disassociation. Carabid larvae, Bathyphantes and total numbers of Linyphiidae showed a strong correlation with weed cover for two of the sample dates. Cereal aphids were disassociated from weed cover on three sampling occasions.

Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference.

Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.

Detection of the virulent form of AVR3a from Phytophthora infestans following artificial evolution of potato resistance gene R3a.

Engineering resistance genes to gain effector recognition is emerging as an important step in attaining broad, durable resistance. We engineered potato resistance gene R3a to gain recognition of the virulent AVR3aEM effector form of Phytophthora infestans. Random mutagenesis, gene shuffling and site-directed mutagenesis of R3a were conducted to produce R3a* variants with gain of recognition towards AVR3aEM. Programmed cell death following gain of recognition was enhanced in iterative rounds of artificial evolution and neared levels observed for recognition of AVR3aKI by R3a. We demonstrated that R3a*-mediated recognition responses, like for R3a, are dependent on SGT1 and HSP90. In addition, this gain of response is associated with re-localisation of R3a* variants from the cytoplasm to late endosomes when co-expressed with either AVR3aKI or AVR3aEM a mechanism that was previously only seen for R3a upon co-infiltration with AVR3aKI. Similarly, AVR3aEM specifically re-localised to the same vesicles upon recognition by R3a* variants, but not with R3a. R3a and R3a* provide resistance to P. infestans isolates expressing AVR3aKI but not those homozygous for AVR3aEM.

Monoclonal antibodies reveal changes in predator efficiency with prey spatial pattern

Spatially explicit predator–prey interactions can alter the predatory potential of natural enemies augmented through conservation biological control. To test hypotheses regarding such interactions and predatory efficiency, we used a combination of molecular techniques and mark–release–recapture to study the foraging behaviour of a generalist carabid predator, Poecilus cupreus, in response to spatial patterns of its cereal aphid prey (Metapolophium dirhodum and Sitobion avenae). Beetle and aphid numbers were measured across two grids of sampling locations, within which aphid spatial pattern had been manipulated to generate patchy and more homogenous distributions. Aphid consumption was measured by enzyme‐linked immunosorbent assays (ELISA) of beetle gut contents, using an aphid‐specific monoclonal antibody. Movement and distribution patterns suggest that P. cupreus does not aggregate at, nor instigate prey‐taxis within, aphid patches. However, more than two‐thirds of the 2169 P. cupreus tested by ELISA had consumed aphids and the proportion of beetles containing aphid proteins was positively related to aphid density. Against expectation, the proportion of predators feeding on aphids was greatest where prey were homogenously distributed, and this was attributed to the loss of partial refuges for prey in aphid patches. The functional value of this type of uniform foraging strategy is ideally suited to early colonization of the crop habitat, when aphid numbers are low, before populations build up and form strong spatial patterns.

Large-Scale Temporal Changes in Spatial Pattern During Declines of Abundance and Occupancy in a Common Moth

We examined the changes in spatial pattern that accompanied the population decline of the garden tiger moth, Arctia caja (L.) in Britain between 1968 and 1999 using Spatial Analysis by Distance Indices (SADIE) techniques. A principal co-ordinate analysis of all pair-wise spatial associations between years indicated three groupings of years: 1969–1978,1979–1990,1991–1999, which revealed three phases during the population decline: an early period with highly structured spatial pattern; a middle period with nearly random distribution overall; and a recent period with highly structured spatial pattern but small abundance overall. The change in spatial structure in the early 1980’s accompanied rapid changes in abundance but preceded a sharp decline in occupancy and confirms that the sharp decline in abundance included decreases from widespread high-density sites. Perhaps unusually, A. caja varied from spatially aggregated to randomly distributed and back to spatially aggregated, all while its abundance declined sharply. Present distribution pattern may reflect past abundance changes poorly in this species. Areas showing the greatest variation in abundance displayed the greatest range in spatial structure, but also the greatest stability of spatial pattern, indicating changes between extremes of spatial pattern occurred slowly. SADIE techniques are a powerful method to quantify temporal changes in spatial pattern and relate them to temporal changes in abundance.

Modifying glycoalkaloid content in transgenic potato - Metabolome impacts

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

The role of large-scale spatially explicit and small-scale localized processes on the population dynamics of cereal aphids

A field-scale study of the spatially explicit interaction between the carabid Poecilus cupreus Linnaeus, and two common aphid species (Sitobion avenae (Fabricius) and Metopolophium dirhodum (Walker)) in winter wheat was conducted. All three species showed considerable spatial pattern at the field scale. Activity-density of P. cupreus was an order of magnitude higher in the central part of the field compared to its periphery. Where P. cupreus activity-density was highest, S. avenae and M. dirhodum population peaks were delayed. Additionally, in the case of M. dirhodum, lower maximum counts were evident where P. cupreus activity-density was highest. An analysis of the movement of individual P. cupreus using release-recapture indicated that those beetles within the centre of the field exhibited reduced displacement, which may have caused the generation or maintenance of spatial pattern. Crop density was also measured throughout the field. Although crop density had no large-scale spatial pattern, its variability at the small-scale was consistent with an influence on aphid population dynamics. This study demonstrates empirically that both large-scale spatially explicit and small-scale localized processes influenced aphid population dynamics simultaneously.

The spatial distribution of canopy-resident and ground-resident cereal aphids (Sitobion avenae and Metopolophium dirhodum) in winter wheat

We investigated, within two cereal fields in Southern England, the within-canopy spatial distribution of the aphids Sitobion avenae and Metopolophium dirhodum in relation to crop yield and plant nitrogen. We extended the study to investigate the spatial distribution of aphids that fell to, or returned from, the ground in order to estimate availability of the within-canopy aphid population to ground-active predators. We revealed that crop canopy aphid spatial pattern was associated with nitrogen or yield. Differences were evident between species: S. avenae was generally negatively associated with yield or plant nitrogen, whilst M. dirhodum exhibited positive association. For both aphid species, we observed strong spatial pattern for aphids falling to the ground and conclude that this could, in part, mediate the effectiveness of ground-active predators as pest control agents.

A method for rapidly mass laser-marking individually coded ground beetles (Coleoptera: Carabidae) in the field

Abstract.  1. A laser‐marking technique is described that may be used to rapidly mass‐mark and individually code insects in the field. In this study, ground beetles were marked but the method is applicable to a wide range of organisms with a hard exoskeleton or shell.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.