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No description available.

No description available.

Bt toxins, and in particular 5 endotoxin Cry toxins, are the bio-insecticides of choice in the control insect pests of agriculture. The emergence of resistance to these toxins has necessitated research for alternative novel bio-insecticides. P. luminescens is an entomopathogenic bacteria symbiotically associated with Heterohabditid nematodes. These nematodes actively seek out insects in the soil. Upon invasion of the insect the nematode regurgitates P. luminescens bacteria, which in turn releases a range of virulence factors which aid in killing and bio-converting the insect host. One of the dominant toxins are high the molecular weight Toxin complexes (Tc's) which are currently under investigation as an alternatives to Bt.

Much of the present knowledge of the activity of pyrethroids on the insect Na⁺ channel has come from the electrophysiological investigation of Na⁺ channels expressed heterologously in the Xenopus laevis oocyte expression system. The effects of the naturally occurring pyrethroid resistance mutations, located within the Na⁺ channel gene(s), have also been studied in this way. These studies have yielded a wealth of information, but make several assumptions - that insect Na⁺ channels behave normally in this alien environment and that the expressed channels accurately represent the range channels present in neurons.

Pyrethroid insecticides remain one of the most widely used classes of insecticides yet resistance threatens their effective continued use. Recently efforts have been applied to understand the genetics and physiology of known mechanisms of resistance. Key mutations of the insect voltage gated sodium channel, such as L1014F, have been implicated in conferring resistance in a number of pest insect species. Two other substitutions of the Ll014 locus have also been reported in the field, l1014S and l1014H. In this study L1014 modified Drosophila para VGSCs were expressed in Xenopus oocytes and their properties examined electrophysiologically using two-electrode voltage-clamp. This revealed significant depolarising shifts in the half activation voltage (V50.act ) from -17.3 mV (wild-type) to -13.1 mV and -13.5 mV for L1014F and L1014H respect ively. whereas the l1014S mutation caused no significant shift in V50.act but its current decayed significantly faster than the wild-type channel. Treatment of the wildtype channel with deltamethrin (>1 nM), permethrin (>30 nM) or DichloroDiphenyl- Trichloroethane (DDT) (>1 uM) resulted in hyperpolarizing shifts in V50.act. Deltamethrin, permethrin and DDT also produced "tail currents" with EC50S of 0.043, 0.40 and 61 uM and maximum modifications of 825, 326 and 6% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V50.act. We conclude that L1014H and L1014S may have arisen through continued exposure to specific pyrethroids and DDT respectively.

Phloem feeding insects (aphids, hoppers) are an important group of pests. They cause damage to their host plant not only by removing nutrients, but also more importantly by transmitting viral diseases. To date, there are no effective long-term control methods for managing the populations of these pests. To address this problem recent research has focused on studying endogenous defence mechanisms, with the aim of their future exploitation in crop protection. This project has studied both sides of plant-insect interactions (plant responses and insect means for invasion), using as a model system Oryza sativa (rice) as the host plant and Nilaparvata lugens (Brown planthopper) as the insect pest. The overall objective of the project was to investigate the molecular and biochemical nature of the 0. sativa - N. lugens interaction as a means of providing the basis for developing novel strategies for control of homopteran pests. For this purpose, insect feeding as the major determinant of host plant responses was studied with particular emphasis on the role of insect saliva, since recent studies on chewing insects have demonstrated that saliva is the main factor modulating host responses. Studying saliva from phloem feeding insects is extremely difficult and to date no salivary compounds from this insect order have been identified within host plants. Hydrogen peroxide (HzOz) is a part of the Reactive Oxygen Species (ROS) system, which includes superoxide (Oy), and hydroxyl radical (OH) as well. Several roles for HzOz have been assigned, such as signalling, cell wall modification and direct toxicity to pathogens and insects. Catalase is the main enzyme responsible for decomposition of hydrogen peroxide (HzOz). Using PCR with degenerate primers, a catalase cDNA (Kat-1) from Nilaparvata lugens salivary glands was identified. Kat-1 transcript abundance was higher in the gut than in salivary glands. Recombinant catalase (~57 kDa) was produced in E. coli, purified and used to produce antibodies in rats. lmmunodetection analysis indicated that there are three possible catalases with molecular masses of ~ 57 kDa, ~70 kDa and ~1 00 kDa in the salivary glands but only one ~57 kDa in the gut. A ~70 kDa immunoreactive protein was detected as well in infested host tissue from the resistant variety, suggesting that catalase is secreted from saliva into host plant tissue. Thus, the role of Nilaparvata lugens salivary catalase may be disruption of signalling and detoxification as well as conditioning of the host cells. Production of ROS occurs under both stressed and non-stressed conditions and ROS concentrations are modulated by enzymatic and non-enzymatic components within the antioxidant system. Key enzymes from the antioxidant system include superoxide dismutase (SOD; EC 1.15.1.1), catalases (CAT; EC 1.11.1.6), ascorbate peroxidases (AsPOX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2) and class III peroxidases (POX; EC 1.11.1.7). The effects of Nilaparvata lugens infestation and saliva on the antioxidant enzyme system in resistant and susceptible varieties were studied by assaying enzyme activity. Temporal and spatial analysis indicated different patterns of enzyme activation in susceptible and resistant host varieties, suggesting that the antioxidant enzyme system is controlled differently in the two rice varieties. At systemic level during infestation, increase of the HzOz-detoxifying system activity (SOD, CAT and AsPOX) was demonstrated only in the resistant variety. Further, saliva induced GR and AsPOX activity in the resistant variety only. These two enzymes are part of the AA-GSH (Ascorbate- Glutathione) cycle, the most important HzOz-detoxifying system in the chloroplasts, peroxisomes and mitochondria. At the mRNA level, there were local differential effects of infestation and saliva in the susceptible and resistant varieties on the expression of SOD and CatA genes. Variation in the expression patterns of genes coding for different forms of SOD, as well as CatA, was demonstrated only in the susceptible variety. Systemic expression of Nilaparvata lugens saliva-responsive genes was studied through subtractive hybridisation and Real Time RT-PCR confirmed the differential expression of several genes. Functional analyses indicated that Nilaparvata lugens saliva altered expression of genes involved In primary metabolism, signalling, transport, translation and regulation and with unknown functions. More than one third of up-regulated genes were implicated in senescence, including biosynthesis of exported amino acids and transport.

This project focuses on the cellular response in insects to the presence of azadirachtin.  Using the two insect cell lines Sf9, <i>Spodoptera frugiperda </i>and a <i>Drosophila </i>cell line Kc167 it was shown that azadirachtin inhibits cell proliferation at EC₅₀’s of 1.38 x 10^-9 M and 1.72 x 10^-7 M respectively.  In contrast azadirachtin had no effect on the Chinese hamster ovarian cell line RR-CHOKI and in the Chinese hamster lung cell line V79-4 where effects were seen only at 10^-3M and above.  Cytotoxicity was observed by visual analysis of single strand DNA in the two insect cell lines with 50% damage being observed after 12 h at 2.2 x 10^-9 M and 3.09 x 10^-8 M for Sf9 and Kc167 cells respectively.  The effects of azadirachtin on protein expression resulted in 52% of Sf9 proteins being down regulated, 29% showing increased expression and 19% showing no response to azadirachtin treatment.  In addition using phage display techniques a monoclonal antibody to azadirachtin was raised.  In terms of functionality the clone selected bound to the conjugated and free forms of azadirachtin but was relatively insensitive with an IC₅₀ of 116 <span style='font-family:Symbol'>mM.  The protein expression yield was also low at 1.43<span style='font-family:Symbol'>mg/ml.  However, in a bid to produce a monoclonal antibody the azadirachtin compound was modified to include a linker arm to which a carrier protein (BSA) was conjugated.  The modified azadirachtin compound 22’23 dihydro 3-hemiglutarate 1 hydroxy azadirachtin proved an invaluable tool in the final chapter of this work.  Using nuclear extracts from the Kc167 cell line and separating these by size exclusion chromatography and Native electrophoresis the 22’23 dihydro 3-hemiglutarate 1 hydroxy azadirachtin - BSA conjugate was used in alliance with a BSA antibody to detect possible azadirachtin binding proteins when present on nitrocellulose membrane.  The characterisation of a protein was established as the <i>Drosophila melanogaster </i>heat shock protein 60 (CG12101-PA).

No description available.

This thesis investigates the potential of two novel insecticides for the control of nuisance Diptera emanating from sewage biological filters. The efficacy of the insect growth regulator pyriproxyfen and a biological nematode Steinernema feltiae for the control of sewage flies were assessed. Both insecticides targeted primarily Sylvicola fenestralis, but also the chironomids Metriocnemus hygropetricus and Limnophyes minimus. Initial laboratory studies determined that concentrations of pyriproxyfen greater than 0.2 mg l-1 inhibited insect egg development or hatching and survival to the first larval instar. Four trials, assessing the efficacy of pyriproxyfen as the formulation Sumilarvoo were undertaken. Significant (P < 0.05) reductions in the emergence of S. fenestralis adults were achieved for doses of 0.5mg L-1, 0.22mg L-1 and 0.2mg L-1 but not for 0.4mg L-1. Reductions ranged from 38% to 100%. There is evidence to suggest that pyriproxyfen may affect adult control over two generations but it may have a limited window of use when targeting adult control. The Chironomid L. minimus was also effectively controlled (P < 0.05) with up to 70% suppression of adult emergence detected. High performance liquid chromatography (HPLC) was ascertained to be an effective analyses tool in determining that pyriproxyfen has a high affinity for sewage sludge's and concentrations > 100 mg L-1 inhibited aerobic and anaerobic purification processes. Laboratory based pathogenicity tests determined that the nematode Steinernema feltiae is a viable biocide capable of infecting the larvae of sewage flies. Field trials at three sites using a dose of one million nematodes per m2 produced significant reductions (P<0.05) in the emergence of S. fenestralis and M hygropetricus. Reductions ranged from 25-67% and 25-91% respectively. No adverse effects on the non-dipteran filter fauna, the purification efficiency, or the invertebrates in the river receiving the work's effluent, were detected after use of both insecticides.