No description available.

Bacillus thuringiensis (Bt) toxins expressed in commercial transgenic crop varieties are all δ-endotoxins (Cry toxins) but the identification of novel vegetative insecticidal proteins (Vip toxins) has extended the range of insecticidal proteins derived from Bt. One such Vip toxin, Vip3A, primarily targets the midgut epithelium cells of susceptible insects as Cry toxins do, although they appear to have different binding sites. The present study investigated the comparative toxicity of Vip3A, Cry1Ab and Cry1Ac against Heliothis virescens (tobacco budworm) and the impact of antibiotics on Bt insecticidal activity. The selection of a resistant Vip3A population led to the determination of cross-resistance, the genetics of resistance and fitness effects. There was very little variability in the natural susceptibility to Vip3A, Cry1Ab and Cry1Ac in the populations tested, although the toxicity of Vip3A was much lower compared to the Cry1A toxins. A Vip3A resistant population was successfully established within 13 selected generations, with little or no cross-resistance to Cry1Ab or Cry1Ac. The inheritance of resistance ranged from almost completely recessive to incompletely dominant with a possible paternal influence, was polygenic and relatively stable. Vip3A resistance showed a fitness benefit, reduced larval development time, and fitness costs, including survival to adult eclosion, reduced egg viability and reduced male mating success. The effects of antibiotics on H. virescens larval susceptibility to Bt toxins varied depending on antibiotic treatment, the Bt toxin used and the larval instar tested. Bt cotton expressing both Vip3A and Cry1Ab to provide activity against a wide range of pest Lepidoptera, including H. virescens, a major cotton pest in the USA is in the process of commercialisation. The present work will help to support a suitable insecticide resistance management strategy for continued use of Bt toxin in transgenic crops.

Insecticide resistance is an important aspect of pest control on both crop pests and vectors of animal diseases. Resistance can be caused by a number of mechanisms, one of which is enhanced detoxification of the insecticide by metabolic enzymes. Synergists may be used in insecticide formulations to inhibit metabolic defences in the insect, allowing the insecticide to reach its target site and kill the insect, thus enhancing the effect of the insecticide. This PhD project investigated the use of the synergist piperonyl butoxide (PBO) in combination with natural pyrethrins (tank mix) and as a pre-treatment prior to application of pyrethrins, as methods of enhancing the efficacy of the insecticide. The insects studied were Myzus persicae, Bemisia tabaci and Musca domestica. Results showed that the combination treatment (tank mix) was at least as good as, and sometimes better than, the pre-treatment. This is unlike the situation for synthetic pyrethroids where pre-treatments have been shown to be more effective than tank mixes. It is proposed that for natural pyrethrins, PBO aids the penetration of the pyrethrins into the insect, and this enhancement effect is greater than full inhibition of the metabolic enzymes. In some cases, the tank mix enabled less insecticide to be used to achieve 50% mortality in resistant insects, compared to a susceptible population treated with pyrethrins alone. A novel laboratory assay was developed to enable the screening of botanical extracts for their ability to inhibit esterase enzymes. This was used to test a range of compounds and those showing esterase inhibition were also screened for their ability to inhibit cytochrome P450 activity. The competency of some of these compounds as synergists was also tested in vivo with some showing potential activity both in vitro and in vivo.

Aedes aegypti is the main vector of the virus that causes dengue fever and its more severe form dengue hemorrhagic fever. As traditional control methods have been unable to prevent its global re-emergence as a significant threat to human health, the development of new control methods is gaining importance. One possibility is a genetics-based control strategy modelled on the traditional sterile insect technique (SIT). The RIDL system (Release of Insects carrying a Dominant Lethal) is such an approach, and has been engineered in Ae. aegypti with tetracycline-dependent repression of a dominant lethal gene construct. This thesis examines some of the aspects of Aedes aegypti mating ecology and behaviour that are relevant for the implementation of SIT-based control programmes, focusing on the competitive fitness of the genetically modified males. The transformed mosquitoes differed from unmodified mosquitoes with a similar genetic background with regard to several life history traits. Though the modified mosquitoes pupated earlier - which may be useful in the mass-rearing of such insects - most of the differences suggest reduced competitive performance of the modified males. These included reduced larval survival, adult longevity, insemination capacity and flight ability. In caged mating trials the modified males were less competitive than their wild type counterparts in direct competition for females. Genetically modified mosquitoes were generally smaller than unmodified mosquitoes reared at high larval densities, highlighting the value of optimising rearing conditions as females preferentially selected larger males for mating. Females exhibited no propensity to re-mate over several gonotrophic cycles, unless they had been mated to sperm-depleted males, in which case secondary insemination was common. In conclusion, genetic manipulation reduced the performance of mosquitoes. However, their competitive disadvantage could be compensated for by high over-flooding ratios upon release. Therefore, accurate estimates of competitive ability, as presented in this thesis, are essential if this control strategy is to prove successful.

The rate of insect development (phenology) is strongly associated with temperature. Within the biological literature, phenologies are estimated largely on the basis of sparsely located point meterological data. The significance of incorporating a geographical dimension was explored in two application areas where phenologies are used, pest risk assessment (PRA) and integrated pest management (IPM). Colorado beetle (<I>leptinotarsa decemlineata)</I> and codling moth (<I>Cydia pomonella)</I> were used as representative non-indigenous and indigenous test organisms. To ensure relevance to both pest risk assessment and integrated pest management applications, phenology models were run using daily meterological data throughout England and Wales. Interpolation was chosen as an efficient means to create spatial temperature 'surfaces' from distributed daily maximum and minimum temperature data observed at a subset of 174 meteorological stations. Because insect pests are known to be highly sensitive to temperature, considerable attention was paid to minimising the errors generated as part of this process relative to that in previous applied agricultural studies. Comparisons between the commonly used trend surface and inverse distance weighting methods of interpolation were made with partial thin plate splines and ordinary kriging. Unlike earlier work, automatic parameter selection was used to calibrate all the interpolation techniques and care was taken to ensure the comparability of estimated temperature values. Error in estimates by all methods was reduced using a number of guiding topo-climate and land cover covariates. The most favourable estimates of maximum and minimum temperatures throughout the country and over the annual cycle were partial thin plate splines, with daily average r.m.s. accuracies computed using jack-knife cross-validation of 0.8°C and 1.13°C respectively. Partial thin plate splines were also found to be more computationally efficient than both inverse distance weighting and de-trended ordinary kriging. This use of jack-knife cross-validation was assessed using a fully independent data set of a further 100 data points, and was found to be statistically comparable. Providing the interaction between phenology models and sequences of geographically relevant temperature data at this daily step and national coverage necessitated the construction of tailor made research software for the project. The coupled temperature interpolation/phenology modelling system was used to provide a range of outputs to explore the accuracy of predicted phenologies over space and time.

No description available.

The insecticide synergist, piperonyl butoxide (PBO), has been used to reduce resistance factors and to characterise metabolic resistance resulting from mixed function oxidases (MFOs). Studies have also shown that PBO can interact with resistance-associated esterases. It is well-documented that the mechanism by which PBO inhibits P450s is the interaction of the methylenedioxyphenyl (MDP) moiety of PBO molecule with the haem moiety of the enzyme. To investigate the interactions between PBO and esterases, a structure activity relationship (SAR) study was carried out using analogues of PBO and E4, a resistanceassociated esterase from the peach-potato aphid Myzus persicae. Results indicate that the polyether and the alkyl chain were fundamentally important in this interaction. Removing one oxygen atom from the MDP moiety of PBO did not affect the binding affinity, but increased affinities resulted from the replacement of the polyether with an alkynyl ether chain or by increasing the length of the alkyl chain. An analogue, EN 16/5-1, that retains the ability to interact with esterases but loses the ability to act on P450s, due to a modification to the MDP moiety, was used in conjunction with PBO to facilitate characterisation of metabolic resistance in M. persicae and pollen beetle Meligethes aeneus. Selected analogues of PBO containing the alkynyl ether side chain exhibited high synergistic effects in vivo and were found to be potent inhibitors of O-deethylation of 7-ethoxycoumarin when tested against pyrethroid resistant M. aeneus. The capability of some of the analogues to act as insecticide synergists was investigated in vivo against two M. persicae clones possessing different metabolic resistance profiles. Finally, the potential of PBO analogues to provide potent and/or specific synergism to overcome insecticide resistance and reduce insecticide titre applied to crops is considered.

Field margins were implemented in UK agri-environment schemes with the aim to increase farmland biodiversity. Recently aerially dispersing aphid enemies have been shown to provide the majority of aphid control in winter wheat fields but there is a lack of research conducted on the aerial predator guild. This thesis examines the effect field margins have on aphid predators at the single field scale, the landscape scale and, using results from a marking study, examines the direct use of a pollen and nectar rich field margin by Episyrphus balteatus. At the single field scale, field margins had a positive effect of the numbers of Cantharidae, Empididae, Linyphiidae and Tachyporus spp. in fields with sown margins compared to those without during wheat growth and total aphid predator numbers were significantly higher in fields with margin surrounds in early May but not later in the year. At the landscape scale, results from twelve winter wheat fields with varying densities of surrounding field margins showed predatory Tachyporus spp. to exhibit a positive correlation at scales above 500m radius and Cantharidae to exhibit a negative correlation at local scales. Implications for field margins exerting both positive and negative influences on the presence of aerially dispersing aphid predators in winter wheat fields are discussed. A marking study using rubidium chloride proved direct utilisation of a pollen and nectar rich field margin by the Syrphid Episyrphus balteatus, and the traps used in this study also highlighted the association in distributions between Empididae and cereal aphids. Overall it was concluded that the presence of field margins does have an effect on the spatial and temporal distributions of some aerially dispersing aphid predators, but the response of each predator group varies depending on numerous interlinking components of their life history and directions for future research are discussed.

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression post-transcriptionally, by binding to specific mRNAs. It has been suggested that miRNAs have a role in "canalising" development and reducing variability in gene expression. Using conditional deletions of the miRNA biogenesis enzyme Dicer in mice, I investigated the effect of miRNA depletion on gene expression during T-cell development. I used flow cytometry to obtain the distribution of proteins on a single cell basis. Proteins encoded by miRNA-regulated transcripts showed a Dicer-dependent increase in both mean protein expression and cell-cell variation. In particular, the genes Sca-1 and Cd44 show increased expression and cell-cell variation in Dicer-deleted double positive thymocytes, and Cd69 showed increased expression and cell-cell variation in Dicer-deleted thymocytes activated by stimulation of the T-cell receptor. Using fluorescent reporter constructs, the effect of the 3’ UTR of each mRNA on reporter expression was investigated to find miRNA binding sites. I identified binding sites for the miR-181, miR-130 and miR-20 miRNA families in the Cd69 3’ untranslated region (UTR). To further investigate how these miRNAs might regulate the expression of Cd69, I investigated how expression of miRNAs changed on T-cell activation: observing that the miR-181 family is downregulated after activation in thymocytes, and the miR-20 family is upregulated after activation in both thymocytes and mature peripheral T-cells. I used both miRNA inhibitors and deletion of specific miRNA families to confirm that the miR-181 and miR-20 families both regulate expression of Cd69 during thymocyte activation. Finally, I looked at theoretical models of how miRNAs might regulate biological noise, and found that the feedforward loop motif can reduce noise compared to an unregulated gene under conditions of moderate miRNA repression. These results show that depletion of miRNAs can result in increased cell-cell variation in developmentally regulated thymocyte genes.

No description available.