Insecticide resistance is a global problem. Insecticide resistance management is very important, considering the time, effort, and cost of discovering and developing a new insecticide. There are diverse resistance mechanisms, but enhanced detoxification through overexpression of cytochrome P450s and target site insensitivity through mutation in insecticide binding site are the two most common mechanisms. The xenobiotic detoxification is divided into three successive phases (I, II and III), which ensures the metabolism and excretion of the detrimental toxins. Each phase comprises of a specific group of metabolizing enzymes such as P450s (phase I), GSTs (phase II) and ABC transporters (phase III). The major goal of my research was to understand the molecular mechanism of insecticide resistance in two economically important coleopteran pests, Leptinotarsa decemlineata and Tribolium castaneum. The transcriptional regulation of the P450 genes mediating insecticide resistance in L. decemlineata (imidacloprid-resistant) and T. castaneum (deltamethrin-resistant) were studied and the xenobiotic trans and cis-elements identified. RNA interference (RNAi), and reporter assays revealed that the cytochrome P450 genes involved in insecticide resistance are regulated by transcription factor Cap n Collar ‘CncC’ and muscle aponeurosis fibromatosis ‘Maf’ belonging to the b-ZIP transcription factor family. Site-directed mutagenesis was employed to identify the binding site for CncC and Maf. Sequencing of RNA isolated from CncC knockdown T. castaneum identified genes regulated by CncC and involved in insecticide detoxification. RNAi and insecticide bioassays confirmed the function of select phase II (glutathione-S-transferases) and phase III (ABC transporters) identified by RNA sequencing. Overall, these data revealed that the xenobiotic transcription factor CncC is the master regulator of multiple genes that are involved in insecticide resistance.

DDT, an extremely widely used organochlorine pesticide, was banned in most developed countries more than 30 years ago. However, DDT residues, including 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), still persist in the environment and have been identified as priority pollutants due to their toxicity and their ability to bioaccumulate and biomagnify in the food chain. In particular, DDE was long believed to be &quotenon-biodegradable&quote, however some microorganisms have now been isolated that are able to metabolise DDE in pure culture. Terrabacter sp. strain DDE-1 was enriched from a DDT-contaminated agricultural soil from the Canterbury plains and is able to metabolise DDE to 4-chlorobenzoic acid when induced with biphenyl. The primary objective of this study was to identify the gene(s) responsible for Terrabacter sp. strain DDE-1�s ability to metabolise DDE and, in particular, to investigate the hypothesis that DDE-1 degrades DDE cometabolically via a biphenyl degradation pathway. Catabolism of biphenyl by strain DDE-1 was demonstrated, and a biphenyl degradation (bph) gene cluster containing bphDA1A2A3A4BCST genes was identified. The bphDA1A2A3A4BC genes are predicted to encode a biphenyl degradation upper pathway for the degradation of biphenyl to benzoate and cis-2-hydroxypenta-2,4-dienoate and the bphST genes are predicted to encode a two-component signal transduction system involved in regulation of biphenyl catabolism. The bph gene cluster was found to be located on a linear plasmid, designated pBPH1. A plasmid-cured strain (MJ-2) was unable to catabolise both biphenyl and DDE, supporting the hypothesis that strain DDE-1 degrades DDE cometabolically via the biphenyl degradation pathway. Furthermore, preliminary evidence from DDE overlayer agar plate assays suggested that Pseudomonas aeruginosa carrying the strain DDE-1 bphA1A2A3A4BC genes is able to catabolise DDE when grown in the presence of biphenyl. A second objective of this study was to characterise pBPH1. The complete 84,054-bp sequence of the plasmid was determined. Annotation of the DNA sequence data revealed seventy-six ORFs predicted to encode proteins, four pseudogenes, and ten gene fragments. Putative functions were assigned to forty-two of the ORF and pseudogenes. Besides biphenyl catabolism, the major functional classes of the predicted proteins were transposition, regulation, heavy metal transport/resistance, and plasmid maintenance and replication. It was shown that pBPH1 has the terminal structural features of an actinomycete invertron, including terminal proteins and terminal inverted repeats (TIRs). This is the first report detailing the nucleotide sequence and characterisation of a (linear) plasmid from the genus Terrabacter.

L'objectif de ce projet était de déterminer les effets létaux et sub-létaux d'un savon insecticide sur le puceron vert du pêcher (Myzus persicae (Sulzer)) et une guêpe parasitoïde du puceron, Aphidius colemani (Viereck). Dans cette étude, les hypothèses suivantes ont été vérifiées: 1) la mortalité des insectes augmente en fonction de la concentration du savon, 2) les doses sub-létales du savon insecticide ont des effets sur la valeur adaptative du puceron (mesurée par la longévité, le taux de développement et la fécondité) et sur la valeur adaptative (estimée par la longueur de tibias et le nombre d'oeufs) et le comportement du parasitoïde (contact antennaire, attaque et ponte). La concentration de savon causant 100% de mortalité 24h après le traitement chez tous les stades du puceron était de 37.5 g/L. La CL₅₀ était de 1.50, 3.25 et de 5.50 g/L pour les larves de stades 1 et 2, 3 et 4, et les adultes respectivement. La longévité des pucerons qui ont survécu à la CL₅₀ était significativement différente de celle des témoins. Par contre, les larves exposées au savon n'ont pas eu un taux de développement significativement différent des témoins. La fécondité des adultes n'était pas significativement différente des témoins. Ce savon insecticide pourrait être une bonne façon de contrôler les pucerons. La concentration de savon causant 100% de mortalité 24h après le traitement chez les adultes de la guêpe était de 17.5 g/L. La CLs₅₀ était de 2.75 g/L. La survie, la longueur des tibias, le nombre d'oeufs matures par femelle émergeant de pucerons parasités et traités au savon, ainsi que le comportement des guêpes mises en contact avec des pucerons traités n'ont pas été affectés. Par contre, les guêpes ont significativement moins pondu dans les pucerons traités ayant survécu au savon. Il semble évident que les programmes de lutte biologique peuvent être améliorés par des applications de savon insecticide si ces dernières sont faites une journée avant le relâcher de parasitoïdes en serre. Ceci éviterait le contact des parasitoïdes avec le savon et permettrait aux pucerons traités de muer. Les parasitoïdes pourront ainsi pondre dans les pucerons qui auront survécu au savon ce qui augmentera le contrôle du puceron.

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for control as they are effective and relatively inexpensive; however, resistance threatens sustainability because alternative insecticides are often more expensive or less effective. Pyrethroid resistance has been identified since 1990 and monitoring has utilized cypermethrin in the adult vial test, but resistance mechanisms have not yet been elucidated at the molecular level. Here we examined field-collected H. zea males resistant to cypermethrin for target site and metabolic resistance mechanisms. We report the cDNA sequence of the H. zea sodium channel a-subunit homologous to the Drosophila para gene and identified known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA-editing. We identified other specimens with significantly higher transcriptional expression levels of cytochrome P450 genes CYP6B8 and CYP6B9 compared to the susceptible, ranging from a factor of 3.7 to 34.9 and 5.6 to 39.6, respectively. In addition, we investigated if differences in insect growth stage and pyrethroid structure affect our ability to predict resistance in the adult vial test. Vial bioassays with cypermethrin, esfenvalerate, and bifenthrin were conducted on third instars and male moths from a susceptible laboratory colony and the F1 generation of a resistant field population. For the resistant population, vial assays using either growth stage gave similar resistance ratios for each of the three pyrethroids, respectively, proving the adult vial test accurately reflects larval resistance. However, resistance ratios varied considerably depending on the pyrethroid used, so values obtained with one pyrethroid may not be predictive of another. This dissertation is the first to identify molecular mechanisms associated with H. zea pyrethroid resistance. Our results suggest carefully chosen pyrethroid structures diagnostic for specific resistance mechanisms could improve regional monitoring programs and development of high throughput assays to detect the resistance mechanisms used in tandem with traditional monitoring may greatly improve our ability to identify and predict resistance and make better control recommendations.

Thesis (M.S.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 48-50). Also available via World Wide Web.

Insecticide-treated nets (ITN) are an effective tool to reduce the burden of malaria in Africa. To address questions concerning the sustained impact of ITN in the face of changing transmission intensity and spreading insecticide resistance a series of four entomological investigations was undertaken in Tanzania. / First, a longitudinal survey of vector populations at different altitudes was conducted in the Hai District, Kilimanjaro Region, Tanzania. Entomological parameters relevant to malaria transmission were measured. Vector densities and entomological inoculation rates decreased dramatically with increasing altitude. Seasonal variations in the host preference and resting behaviour of Anopheles arabiensismay influence malaria transmission and control. / Second, the insecticide susceptibility status of vector populations from agricultural sites in the Kilimanjaro Region was evaluated. Malaria vectors from six sites were highly susceptible to diagnostic concentrations of pyrethroid insecticides and DDT in susceptibility tests. Permethrin tolerance in populations of An. arabiensis from an irrigated rice-growing area was not directly related to agricultural use of insecticides. Synergist bioassays indicated the contribution of a metabolic oxidase-based mechanism to the observed resistance while the knockdown resistance (kdr) mechanism was not detected. Close monitoring of resistance development in this area is needed. / Third, the biological efficacy of ITN and the insecticide susceptibility status of mosquito populations from areas with long-term use of ITN were investigated. Data were compiled from national surveys and long-term ITN projects to examine trends in resistance development in Tanzania. Anopheles species were highly susceptible to ITN and diagnostic concentrations of pyrethroid insecticides, while 'nuisance-biting' Culex quinquefasciatus displayed low susceptibility to these compounds. Low efficacy of ITN against C. quinquefasciatus has implications for user acceptance of ITN. / Finally, a novel method for kdr genotyping was developed and evaluated. Sequence specific oligonucleotide probes (SSOP) were used to identify kdr mutations in Anopheles gambiae s.l. in a high-throughput enzyme-linked immunosorbent assay (ELISA) format. Evaluation of the method on field-collected specimens detected the leucine-phenylalanine kdr mutation in two individual An. arabiensis, raising implications for the geographic spread of kdr-based resistance. The novel SSOP-ELISA method is a useful tool for high-throughput screening allowing sensitive detection of insecticide resistance.

Populations of Cimex lectularius, the bed bug, have resurged around the world posing significant challenges for pest management professionals and causing physical, economic, and emotional strife. Pyrethroid resistance has been found in the vast majority of populations making pest management more difficult. The objectives of my dissertation research were to document the evolution of resistance to pyrethroid and neonicotinoid combination products (called combination products here) and to a neonicotinoid in the laboratory, to record potential fitness costs to resistance to the combination products, and to compare the efficacy of nine insecticides on six populations. In the laboratory, populations of bed bugs evolve resistance rapidly to a combination product and that resistance translates into cross resistance to another combination product. In a follow up experiment, resistance to a neonicotinoid occurred after three generations of selection. Cross resistance between neonicotinoid and pyrethroid resistance was also found, likely due to a common detoxification mechanism (cytochrome P450 mediated metabolism). Resistance was associated with life history costs in three populations that had been selected with a combination product. Therefore, in the absence of selection pressure, populations of bed bugs should revert towards increasing susceptibility. Two pyrethroid products and three combination products were effective at killing three populations of bed bugs but were relatively ineffective against three other populations. However, the combination product, Transport GHP®, the single action pyrrole product, Phantom SC®, and the single action desiccant, CimeXa®, killed 95 to 100% of all populations investigated over a 14-day exposure. Taken together, results reported in this dissertation suggest that insecticide resistance management may be a useful tool for extending the efficacy of insecticides for control of C. lectularius.

DDT, an extremely widely used organochlorine pesticide, was banned in most developed countries more than 30 years ago. However, DDT residues, including 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), still persist in the environment and have been identified as priority pollutants due to their toxicity and their ability to bioaccumulate and biomagnify in the food chain. In particular, DDE was long believed to be &quotenon-biodegradable&quote, however some microorganisms have now been isolated that are able to metabolise DDE in pure culture. Terrabacter sp. strain DDE-1 was enriched from a DDT-contaminated agricultural soil from the Canterbury plains and is able to metabolise DDE to 4-chlorobenzoic acid when induced with biphenyl. The primary objective of this study was to identify the gene(s) responsible for Terrabacter sp. strain DDE-1�s ability to metabolise DDE and, in particular, to investigate the hypothesis that DDE-1 degrades DDE cometabolically via a biphenyl degradation pathway. Catabolism of biphenyl by strain DDE-1 was demonstrated, and a biphenyl degradation (bph) gene cluster containing bphDA1A2A3A4BCST genes was identified. The bphDA1A2A3A4BC genes are predicted to encode a biphenyl degradation upper pathway for the degradation of biphenyl to benzoate and cis-2-hydroxypenta-2,4-dienoate and the bphST genes are predicted to encode a two-component signal transduction system involved in regulation of biphenyl catabolism. The bph gene cluster was found to be located on a linear plasmid, designated pBPH1. A plasmid-cured strain (MJ-2) was unable to catabolise both biphenyl and DDE, supporting the hypothesis that strain DDE-1 degrades DDE cometabolically via the biphenyl degradation pathway. Furthermore, preliminary evidence from DDE overlayer agar plate assays suggested that Pseudomonas aeruginosa carrying the strain DDE-1 bphA1A2A3A4BC genes is able to catabolise DDE when grown in the presence of biphenyl. A second objective of this study was to characterise pBPH1. The complete 84,054-bp sequence of the plasmid was determined. Annotation of the DNA sequence data revealed seventy-six ORFs predicted to encode proteins, four pseudogenes, and ten gene fragments. Putative functions were assigned to forty-two of the ORF and pseudogenes. Besides biphenyl catabolism, the major functional classes of the predicted proteins were transposition, regulation, heavy metal transport/resistance, and plasmid maintenance and replication. It was shown that pBPH1 has the terminal structural features of an actinomycete invertron, including terminal proteins and terminal inverted repeats (TIRs). This is the first report detailing the nucleotide sequence and characterisation of a (linear) plasmid from the genus Terrabacter.

Thèse (D. en biologie)--Université du Québec à Montréal, 2005. / En tête du titre: Université du Québec à Montréal. Bibliogr.: f. 203-224. Publié aussi en version électronique.

Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2001. / Bibliography: leaves 177-191.