Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Ondrej Tomáš Supervisor: PharmDr. Marie Vopršalová, CSc. Consultant supervisor: mjr. PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: Evaluation of newly prepared insecticides in vitro Malaria is a widespread infection and one of the most dangerous diseases transmitted by insects. It threatens lives of millions of people all around the world, thus its regulation is necessary. Most common malaria vectors are mosquitoes of genus Anopheles. Novel structures of insecticides with selective inhibition of mosquito acetylcholinesterase are subjects of research, with an intention to deal with this problem. The aim of this work was to test six newly prepared succinimide derivatives with insecticidal potential. The ability of these compounds to inhibit Anopheles gambiae mosquito (AgAChE) and human acetylcholinesterase (hAChE) was evaluated. Leading structures of these compounds were also tested to find relations between chemical structure and biological activity. The modified Ellman's method was used to obtain the half maximal inhibitory concentration (IC50) values for both enzymes. Tested substances were able to inhibit only hAChE and none of them displayed activity against AgAChE. Compound...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Ondrej Tomáš Supervisor: PharmDr. Marie Vopršalová, CSc. Consultant supervisor: mjr. PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: Evaluation of newly prepared insecticides in vitro Malaria is a widespread infection and one of the most dangerous diseases transmitted by insects. It threatens lives of millions of people all around the world, thus its regulation is necessary. Most common malaria vectors are mosquitoes of genus Anopheles. Novel structures of insecticides with selective inhibition of mosquito acetylcholinesterase are subjects of research, with an intention to deal with this problem. The aim of this work was to test six newly prepared succinimide derivatives with insecticidal potential. The ability of these compounds to inhibit Anopheles gambiae mosquito (AgAChE) and human acetylcholinesterase (hAChE) was evaluated. Leading structures of these compounds were also tested to find relations between chemical structure and biological activity. The modified Ellman's method was used to obtain the half maximal inhibitory concentration (IC50) values for both enzymes. Tested substances were able to inhibit only hAChE and none of them displayed activity against AgAChE. Compound...

The development of pyrethroid resistance in mosquitoes threatens the goal of malaria elimination in Africa. Alternative insecticides, e.g. organophosphates, can be considered to control pyrethroid resistant mosquitoes. The problem associated with the deployment of organophosphate-based insecticides is their high volatility. Conventional application forms have a fairly short residual efficacy. This study aimed at extending the residual efficacy of an organophosphate insecticide by using a polymer matrix as a slow release device. A multilayer film blower was used to produce a trilayer film. The middle layer comprised poly(ethylene-co-vinyl acetate), i.e. EVA polymer, impregnated with malathion. This was sandwiched by two low density polyethylene (LDPE) outer layers. These acted as semi-permeable membrane-like barriers that slowed down the release of the contact insecticide to the surfaces of the film. In theory, such a film could be deployed as a long-lasting insecticide-treated wall lining in pyrethroid resistant settings. Scanning electron microscopy (SEM) confirmed the trilayer film structure of the blown film. The malathion release from the film was tracked with Fourier transform infrared spectroscopy (FTIR). The malathion absorption band in the FTIR spectra disappeared gradually over time. Confocal Raman analysis showed a malathion concentration gradient across the thickness of the polyethylene layers. These results suggested diffusion-controlled transport through the LDPE membranes. Bioassays indicated that the residual efficacy of the malathion, against mosquitoes, was increased to about six months. This means that trilayer films, impregnated with an organophosphate, may have potential as alternative mosquito control interventions in pyrethroid resistant settings. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

<p>  </p> <p>The objectives of this dissertation included comparing the whole gut bacterial profiles of insecticide resistant and susceptible <em>B. germanica</em> and determining how these profiles, as well as the structure and function of the gut microbiome, change in the presence of an antibiotic. Additional goals were to investigate how antibiotic treatment impacts the toxicity of the bait insecticides fipronil, abamectin and indoxacarb, and to determine how gut bacteria, and specifically the enzymes originating within gut bacteria, metabolize and convert ingested indoxacarb into its toxic metabolite DCJW. Findings show that pre-treatment with the antimicrobial compound kanamycin (KAN) led to reductions in resistance levels for fipronil and abamectin, but also increased basal toxicity levels in both resistant and susceptible strains tested. 16S bacterial sequence surveys revealed that resistant and susceptible cockroach strains were more similar before KAN treatment than after, with a stronger dysbiosis effect in the resistant strain. For the insecticide indoxacarb, regardless of strain, roaches treated with kanamycin-infused water in feeding bioassays were more susceptible compared to the control treatment, but in vial (surface contact) bioassays, only susceptible cockroaches experienced a significant shift in mortality. When the frass of indoxacarb-fed cockroaches was analyzed, fewer molecules of the hydrolytic metabolite DCJW were produced with the introduction of an antibiotic (KAN). This result was further corroborated by esterase activity assays of whole homogenized cockroach guts. All results considered, these findings provide novel evidence of microbe-mediated pro-insecticide activation in the cockroach gut. Overall, the results of this dissertation reveal previously unknown relationships between gut microbiota and their insect hosts. These microbiome relationships exposed important cockroach strain differences which may extend to the host population level. Furthermore, this research has connected a change in enzyme activity in the gut microbiome with indoxacarb, a very important marketplace pro-insecticide.</p>

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.

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is the most important insect pest of cotton, Gossypium hirsutum (L.), in Mississippi. This research project was initiated to gain a better understanding of selected crop production factors that can improve tarnished plant bug integrated pest management. Results suggest that irrigation strategies and varietal pubescence can significantly influence tarnished plant bug management in cotton. Most notably, delaying irrigation for as long as possible and planting hairy varieties can minimize the impact of tarnished plant bug on cotton yields and reduce the number of insecticides needed to manage this pest. Additionally, these results show that tarnished plant bug management is most critical during the first four weeks of flowering. Results from these experiments will be used to improve the current integrated pest management program for tarnished plant bug in cotton and make cotton production more sustainable for Mississippi producers.

No description available.

Chemical control is a critical component of integrated pest management in cotton and soybean production. Residual efficacy of foliar insecticides can be highly variable and difficult to quantify due to several factors. The purpose of this research was to determine residual control and concentrations in flowering structures of commonly used insecticides. This research focused on the concentrations of active ingredient within the plant as well as efficacy over time. Previous research suggested chlorantraniliprole had a long residual and was highly lethal on corn earworm up to 28 days after treatment (DAT) in soybean; however, the results found in cotton were inconclusive. From this research, concentrations of chlorantraniliprole were found in flowering structures of both soybean and cotton up to 14 DAT. Bioassays conducted from chemical concentrations suggest reduced rates of chlorantraniliprole, similar to what was found in the flowering structures, provided mortality of corn earworm up to 64%.

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.

The inheritance of resistance to chlordane in the German cockroach, Blattell germanica. (L.), was studied by determining the toxicity of chlordane to the following strains of roaches: a) chlordane-resistant and non-resistant strains, b)F₁ and F₂ progeny of reciprocal crosses between two parental strains, c) progeny of pair matings within resistant and non-resistant strains, and from a cross between resistant females and non-resistant males. / Ph. D.