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.

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.

Thesis (M.S.)--University of Tennessee, Knoxville, 2008. / Title from title page screen (viewed on Mar. 13, 2009). Thesis advisor: Jerome F. Grant. Vita. Includes bibliographical references.

Thesis (M.S.)--University of Tennessee, Knoxville, 2007. / Title from title page screen (viewed on June 4, 2008). Thesis advisor: Paris L. Lambdin. Vita. Includes bibliographical references.

Dissertation (Ph.D.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:12:53Z : No. of bitstreams: 1 patarro_tf_me_sjrp.pdf: 771112 bytes, checksum: a164331065235e873e875755e608132b (MD5) / A alta incidência da dengue no Brasil, causada pela elevada freqüência de seu vetor, o Aedes aegypti, torna importante conhecer a organização de suas populações em termos de diferenciação genética. Esse conhecimento poderá levar a métodos de monitoramento e controle mais eficientes. No presente trabalho, a técnica RAPD-PCR foi utilizada na análise de quatro populações brasileiras de A. aegypti, sendo três classificadas pela SUCEN (Superintendência do Controle de Endemias) como resistentes aos inseticidas utilizados para seu controle (São Luís- SL; São José do Rio Preto- RP; Araçatuba- AR) e uma classificada como portadora de resistência em desenvolvimento (Bauru- BA). Uma quinta população, procedente dos Estados Unidos, suscetível aos mesmos inseticidas (Rockefeller- RO) foi utilizada para comparação. A aplicação dos métodos estatísticos de Nei, (1973, 1978) produziu índices que permitem considerar o conjunto de populações analisadas, no total de primers, como portador de diferenciação genética muito alta conforme a classificação de Wright (1978). Assim indicaram os valores de Gst (0,277), que mede a diferenciação gênica; de Hs (0,129), que mede a heterozigose média e de Ht (0,181), que mede a heterozigose total. Porém, considerando-se as comparações das populações duas a duas, verifica-se que essa diferenciação foi variável, sendo que a comparação entre as populações RP e AR mostrou que estas são as populações menos diferenciadas geneticamente, enquanto as mais diferenciadas foram SL x RO e SL x BA. Os cálculos de similaridade de Nei e Li (1979) confirmaram os dados obtidos com os índices mencionados, reforçando a idéia de maior similaridade entre RP e AR, seguida de RO e BA. De modo geral, pode-se dizer que não é fácil interpretar a estrutura das populações de A. aegypti quanto às causas de sua diferenciação... / The high incidence of dengue in Brazil, caused by the high frequency of its vector, Aedes aegypti, makes the organization of their populations in terms of genetic differentiation something important to know. This knowledge could lead to more efficient methods of monitoring and control of the vector. In this study, the RAPD-PCR was used in the analysis of four Brazilian populations of A. aegypti, three of which were classified by SUCEN (Superintencia do Controle de Endemias) as resistant to the insecticides used for its control (San Luis-SL; São Jose do Rio Preto, RP; Araçatuba-AR) and one considered in resistance development (Bauru-BA). A fifth population, from the United States, susceptible to these insecticides (Rockefeller-RO) was used for comparison. The application of the statistical methods of Nei (1973, 1978) produced indices that allowed us to consider the set of populations studied, in the total of primers, as having a very high genetic differentiation, according to the classification of Wright (1978). This is indicated by the values of Gst (0.277), which measure the genetic differentiation, the values of Hs (0.129), which measures the average heterozygosity and of Ht (0.181), that measure the total heterozygosity. However, considering the pair-wise comparisons of populations, this differentiation was variable, being the populations RP and AR the less differentiated genetically, while the more differentiated were SL x RO and BA x SL. The estimation of the similarity of Nei and Li (1979) confirmed the data obtained with the indices mentioned, reinforcing the idea of greater similarity between RP and AR, followed by RO and BA. In general, it is not easy to interpret the structure of A. aegypti populations as to on the causes of their genetic differentiation, since they are under strong interference of the human activity, which includes not only the passive transport... (Complete abstract click electronic access below)

Culex mosquitoes are known for carrying several harmful viruses in the United States. Culex tarsalis is found in rural as well as some residential areas in the Western United States, so they are under insecticide pressure from both agricultural spraying and vector control. In response to insecticide pressure, mosquitoes can evolve two primary resistance mechanisms: target site insensitivity, as a result of DNA mutation, and elevated levels of detoxifying enzymes (GST, alpha and beta esterases, and P450 oxidases). The two types of target site insensitivity studied here in Cx. tarsalis are kdr, which is a mutation in the para-type voltage gated sodium channel and ace-1, which is a mutation in acetylcholinesterase gene. This study focused on a population of Cx. tarsalis in Sutter County, where insecticide use shifted from sumithrin to Naled over the course of the summer. The goal of this study was to determine if there was resistance to insecticides and characterize the mechanisms of resistance. Mosquitoes were separated into resistance levels based on CDC bottle bioassay results using Naled, sumithrin, and permethrin insecticides. Mosquitoes were used to test for elevated levels of detoxifying enzymes and genetic qPCR testing for either kdr and ace-1 mutations. Bottle bioassay results suggest Cx. tarsalis populations from Sutter County are mostly resistant to pyrethroids while not being resistant to organophosphates. Enzymatic assays suggest high concentrations or activities of detoxifying enzymes are commonly seen in resistant individuals, occasionally elevated levels of multiple enzymes within an individual. The ace-1 mutation was seen in a single susceptible individual (0.036%). Either one or two kdr alleles were present in every single semi-resistant or resistant mosquito tested.

No description available.

The tobacco-adapted form of the green peach aphid, Myzus persicae (Sulzer), is one of the most important insect pests of tobacco in the United States and around the world. Insecticides play a major role in controlling the aphid on tobacco because natural enemies usually fail to maintain its populations below damaging levels. The aphid has a history of developing resistance to many insecticides. Therefore, baseline information on the aphid's susceptibility to imidacloprid and other insecticides is critical for developing future resistant management programs to minimize losses attributed to the aphid. Studies were conducted on colonies of the tobacco-adapted form of the green peach aphid collected from nine states in the eastern United States in 2004-2007. The susceptibility of 151 colonies to imidacloprid was determined in serial leaf-dip bioassays. When combined over the four years, 18, 14, and 4% of the colonies had 10- to 20-fold, 20- to 30-fold, and 30- to 90-fold resistance ratios, respectively, suggesting that high levels of resistance to imidacloprid are present in field populations of the aphid. A colony collected near Clayton, NC had the highest LC50 value (31 ppm) combined over six tests and three years, but the average resistance ratios for the first three runs was over 130-fold (48 ppm). Geographic location had little effect on susceptibility to imidacloprid. Aphid colonies (136) including red, green, and orange color morphs were screened for total esterase activity using microplate assay with 1-Naphthyl acetate as the substrate. The green morphs generally had lower esterase levels than the red and orange morphs. All orange morphs had among the highest esterase activities. Esterase activities of red and green morphs were positively correlated with LC50 values as determined by leaf-dip bioassays for acephate and methomyl. All 25 colonies tested for esterase gene amplification had either E4 or FE4 gene amplification. The amplification of both E4 and FE4 seen as an 865-bp band characteristic of the FE4 gene and an additional 381-bp band characteristic of a deleted upstream region of the E4 gene occurred in all (4) orange morphs and one (1 of 9) green morph. Target-site insensitivity of acetylcholinesterase (AChE), as modified AChE resistance (MACE) was assessed in 65 colonies of field-collected tobacco-adapted forms of M. persicae. Eight colonies over a range of AChE activity were selected to study inhibition of AChE in the presence of two carbamate insecticides, methomyl and pirimicarb. IC50 values for methomyl ranged from 0.35 to 2.4 μM while six of eight colonies had lower values with a range of 0.16 to 0.30 μM for pirimicarb. Two colonies that were inhibited by methomyl had very high IC50 values of 40.4 and 98.6 μM for pirimicarb. Such insensitivity may be due to mutations in the ace2 gene, but this needs to be confirmed by genetic and molecular analysis. Glutathione S- transferases (GSTs), isoenzymes that are involved in the metabolism and detoxification of many xenobiotic compounds were quantified for 100 colonies by CDNB conjugation. There was a wide range of GST activity for the red (8 to 343 pmol/min/mg protein) and green (15.3 to 330 pmol/min mg protein) morphs, but all six orange morphs collected in 2007 had a narrower range (160 to 211 pmol/min/mg protein). About 45% of the red morphs had GST activity from 200-300 pmol/min/mg of protein, while 53% of the green morphs had a range of 100-200 pmol/min/mg protein. The influence of temperature-mediated synergisms on the toxicity of insecticides in red and green color morphs of the tobacco-adapted from of M. persicae were evaluated using leaf-dip bioassay procedures in laboratory incubators. Post-exposure temperatures of 15, 20, and 25°C were evaluated for four classes of insecticides, acephate, imidacloprid, lambda-cyhalothrin, and methomyl. The temperature change from 15 to 20°C caused almost a 3-fold increase in toxicity to the red and green color morphs for methomyl, acephate, and imidacloprid. In contrast, the toxicity of lambda-cyhalothrin decreased as the temperature increased, showing a negative temperature coefficient. Bioassay experiments conducted with the red morph for indirect estimates of imidacloprid concentrations in flue-cured tobacco showed that leaf position, imidacloprid rate and time after application affected the concentration of the toxicant in the leaf. The differences in aphid mortality between the lower and upper leaf positions indicate that the concentration of imidacloprid and its metabolites were unevenly distributed with the lowest mortality for aphids feeding on the younger, upper leaves and the highest for those feeding on the older, lower leaves. In field experiments, higher aphid populations occurred on tobacco treated with imidacloprid less than the field recommended rate of 41.4 ml/1,000 plants. The development of aphid populations in the field was consistent with the laboratory bioassays. Field trials were conducted to evaluate the performance of various insecticides currently registered for aphid control on tobacco. Imidacloprid applied as a tray drench treatment and acephate as foliar sprays were the most effective treatments. Moderate declines in control with imidacloprid were observed at 75-87 d after transplanting in 2006 and 2007. Aldicarb gave good to excellent control in one of three years, but only fair to poor control in the other two years. Methomyl and lambda-cyhalothrin gave good control in all three years except the residual was shorter. The poor performance of aldicarb in the two years may have been related to the presence of E4 or FE4 resistance in the naturally occurring TGPA in the experimental plots. / Ph. D.