The insecticide dichloro-diphenyl-trichloroethane (DDT) is widely used in indoor residual spraying (IRS) for malaria control owing to its longer residual efficacy compared to other World Health Organization (WHO) alternatives. It was envisaged that by investigating mechanisms of degradation of these alternative insecticides, a better understanding would be obtained on strategies of stabilising them and rendering their efficacy comparable with or better than DDT, hence providing less controversial and more acceptable and effective alternative insecticide formulations to DDT. This study sought to investigate the reasons behind the reported longer lasting behaviour of DDT by exposing all the WHO approved insecticides to high temperature, high humidity and ultra-violet light. Interactions between the insecticides and some mineral powders in the presence of an aqueous medium were also tested. Simple insecticidal paints were made using slurries of these mineral powders whilst some insecticides were dispersed into a conventional acrylic paint binder. These formulations were then spray painted on neat and manure coated mud plaques, representative of the material typically used in rural mud houses, at twice the upper limit of the WHO recommended dosage range. DDT was applied directly onto mud plaques at four times the WHO recommended concentration and on manure plaques at twice WHO recommended concentration. All plaques were subjected to accelerated ageing conditions of 40 °C and a relative humidity of 90%. In the accelerated ageing tests, Fourier transform infra-red (FTIR) interferograms showed that pyrethroids were the most stable insecticides compared to carbamates and DDT. High temperature oxidation, ultra violet light and humidity were ruled out as the cause of failure of the alternative insecticides. Gas chromatography (GC) interferograms showed that phosphogypsum stabilised the insecticides the most against alkaline degradation. Bioassay testing showed that the period of efficacy of these formulations was comparable to that of DDT sprayed at 4 times the upper limit of the WHO recommended dosage range. Bioassay testing also showed that these insecticidal “paint” formulations stabilised the insecticides on cattle manure coated surfaces as compared to DDT sprayed directly on similar surfaces. Bioassay experiments indicated that incorporating insecticides into a conventional paint binder or adsorbing them onto phosphogypsum provided effective life spans, under accelerated ageing conditions, comparable to or exceeding that of DDT directly applied to typical soil substrates. Best results were obtained with propoxur in standard acrylic emulsion paint. Similarly, insecticides adsorbed on phosphogypsum and sprayed on cattle manure coated surfaces provided superior lifespans compared with DDT sprayed directly on a similar surface. Copyright / Dissertation (MEng)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

Juvenile hormone (JH) is the central hormonal regulator of life-history trade-offs in many insects. In Aedes aegypti, JH regulates reproductive development after emergence. Little is known about JH’s physiological functions after reproductive development is complete or JH’s role in mediating life-history trade-offs. By examining the effect of hormones, nutrition, and mating on ovarian physiology during the previtellogenic resting stage, critical roles were determined for these factors in mediating life-history trade-offs and reproductive output. The extent of follicular resorption during the previtellogenic resting stage is dependent on nutritional quality. Feeding females a low quality diet during the resting stage causes the rate of follicular resorption to increase and reproductive output to decrease. Conversely, feeding females a high quality diet causes resorption to remain low. The extent of resorption can be increased by separating the ovaries from a source of JH or decreased by exogenous application of methoprene. Active caspases were localized to resorbing follicles indicating that an apoptosis-like mechanism participates in follicular resorption. Accumulations of neutral lipids and the accumulation of mRNA’s integral to endocytosis and oocyte development such as the vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) were also examined under various nutritional and hormonal conditions. The abundance of mRNA's and neutral lipid content increased within the previtellogenic ovary as mosquitoes were offered increasing sucrose concentrations or were treated with methoprene. These same nutritional and hormonal manipulations altered the extent of resorption after a blood meal indicating that the fate of follicles and overall fecundity depends, in part, on nutritional and hormonal status during the previtellogenic resting stage. Mating female mosquitoes also altered follicle quality and resorption similarly to nutrition or hormonal application and demonstrates that male accessory gland substances such as JH III passed to the female during copulation have a strong effect on ovarian physiology during the previtellogenic resting stage and can influence reproductive output. Taken together these results demonstrate that the previtellogenic resting stage is not an inactive period but is instead a period marked by extensive life-history and fitness trade-offs in response to nutrition, hormones and mating stimuli.

Malaria presents a global health risk that is becoming increasingly difficult to treat due to increased resistance of both the parasite and mosquito to all known drugs. Identification of novel drug targets are therefore essential in the fight against malaria. Polyamines are small flexible polycations that are represented by three basic polyamines. The interaction of polyamines with various macromolecules may lead to stabilisation of DNA, regulation of transcription, replication, and also have an important role in cellular differentiation, proliferation, growth and division. Therefore, disruption of polyamine biosynthesis presents a unique drug target worth exploiting. Polyamine biosynthesis in P. falciparum is regulated by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) complex, which is unique to P. falciparum and differs completely from human polyamine biosyntehsis. The inhibition of AdoMetDC induces spermidine and subsequent spermine depletion within the parasite that ultimately results in cell cycle arrest. A functional genomics approach was used within this study to identify a global response of the parasite due to the inhibition of AdoMetDC with the irreversible inhibitor, MDL73811. The proteomics approach was optimised for conditions specific to our laboratory with regard to protein extraction, Plasmodial protein quantification, spot detection and finally protein identification by mass spectrometry (MS). This methodology resulted in reliable spot detection and achieved a 95% success rate in MS/MS identification of protein spots. Application of this methodology to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots from the Plasmodial ring and trophozoite stages, which also confirmed stage specific protein production. Various protein isoforms were present which may be of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle. Subsequent application of the 2-DE methodology to the proteome of AdoMetDC inhibited parasites resulted in the identification of 61 unique Plasmodial protein groups that were differentially affected by the inhibition of AdoMetDC in 2 time points. The transcriptome of AdoMetDC inhibited parasites were also investigated at 3 time points. Investigation into the transcriptome revealed the differential regulation of 549 transcripts, which included the differential regulation of polyamine specific transcripts. Inhibition of AdoMetDC provided a unique polyamine specific transcriptomic signature profile that demonstrated unique interactions between AdoMetDC inhibition and folate biosynthesis, redox metabolism and cytoskeleton biogenesis. The results presented provide evidence that the parasite responds to AdoMetDC inhibition by the regulation of the transcriptome and proteome in an attempt to alleviate the effects of AdoMetDC inhibition. Further analyses of the metabolome also provided evidence for the tight regulation of the AdoMet cycle. Overall, this study demonstrated important functional consequences as a result of AdoMetDC inhibition. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

Aedes aegypti (Ae. aegypti) is one of the most important vectors of dengue, chikungunya and yellow fever viruses. The use of chemical control strategies such as insecticides is associated with problems including the development of insecticide resistance, side effects on animal and human health, and environmental concerns. Because current methods have not proven sufficient to control these diseases, developing novel, genetics-based, control strategies to limit the transmission of disease is urgently needed. Increased knowledge about mosquito-pathogen relationships and the molecular biology of mosquitoes now makes it possible to generate transgenic mosquito strains that are unable to transmit various parasites or viruses. Ae. aegypti genetic experiments are enabled, and limited by, the catalog of promoter elements available to drive transgene expression. To find a promoter able to drive robust expression of firefly (FF) luciferase in Ae. aegypti embryos, an experiment was designed to compare Ae. aegypti endogenous and exogenous promoters. The PUb promoter was found to be extremely robust in expression of FF luciferase in different stages of embryonic development from 2-72 hours after injection. In subsequent experiments, transformation frequency was calculated using four different promoters (IE1, UbL40, hsp82 and PUb) to express the Mos1 transposase open reading frame in Mos1-mediated transgenesis. Germline transformation efficiency and size of transgenic cluster were not significantly different when using endogenous Ae. aegypti PUb or the commonly used exogenous Drosophila hsp82 promoter to express Mos1 transposase. This study also describes the development of new tools for gene editing in the Ae. aegypti mosquito genome and the use of these tools to design an efficient gene drive system in this mosquito. Homing endonucleases (HEs) are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. The activities of four HEs (Y2-I-AniI, I-CreI, I-PpoI, and I-SceI) were investigated for their ability to catalyze the excision of genomic segments from the Ae. aegypti genome. All four enzymes were found to be active in Ae. aegypti; however, the activity of Y2-I-AniI was higher compared to the other three enzymes. Single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways were identified as mechanisms to repair HE-induced dsDNA breaks. TALE nucleases (TALENs) are a group of artificial enzymes capable of generating site-specific DNA lesions. To examine the ability of TALENs for gene editing in Ae. aegypti, a pair of TALENs targeted to the kmo gene were expressed from a plasmid following embryonic injection. Twenty to forty percent of fertile G0 produced white-eyed progeny which resulted from disruption of the kmo gene. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. A small deletion of one to seven bp occurred at the TALEN recognition site. These results show that TALEN and HEs are highly active in the Ae. aegypti germline and can be used for gene editing and gene drive strategies in Ae. aegypti. / Ph. D.

Bacillus-derived larvicides, which selectively target mosquito (Diptera: Nematocera: Culicidae) populations to reduce nuisance and health risks, were applied in the South March Highlands Conservation Forest near residential neighbourhoods in Ottawa, Ontario. The objective was to assess effects of application on the nontarget mosquito relative, Chironomidae (Diptera: Nematocera: Chironomidae), and other nontarget aquatic taxa captured using emergence traps. A secondary objective was to assess physicochemical variables that influence Chironomidae emergence. Study ponds received an application of Bacillus thuringiensis var. israeliensis, a subset also received an application of Bacillus sphaericus, and a group of control ponds were left untreated over 3 years (2016-2018). Weekly sampling included trap collections and measurements of water temperature, pH, water depth, conductivity, dissolved oxygen, ammonia, nitrate, and sulphate. Drought in 2016, high precipitation throughout 2017, and seasonal precipitation in 2018 influenced variable physicochemical conditions. Principal component analyses identified differences between sampling groups and between years. Redundancy analyses correlated insect emergence with pond pH, average water depth and water temperature and indicated a strong relationship between Chironomidae emergence and average water depth. Although significantly less Chironomidae annual emergence was observed at treated sites in 2017 and 2018, zero-inflated negative binomial generalized linear mixed modelling failed to detect a significant Bti treatment effect when controlling for within group variation. Rather, variations in pH, mean water depth and water temperature were identified as drivers of Chironomidae emergence. Culicidae emergence was reduced to zero briefly following treatment in 2017 and 2018. The model detected a marginal negative treatment effect on Culicidae in 2017 only, and a positive treatment effect in 2018 at the onset of a secondary hydroperiod, in the absence of treatment. Variations in pH and water temperature were also identified to be drivers of Culicidae emergence. Modelling failed to detect treatment effects on any of the nontarget taxa abundance, including Diptera, Lepidoptera, Ephemeroptera, Odonata, Coleoptera, Hymenoptera, and Arachnida. An inverse relationship between insectivore and prey taxa abundance was observed. In 2018, taxa richness increased between years and trended higher at treated sites and a positive relationship between insectivore and prey taxa richness was observed. In 2017, Shannon-Weiner index and Simpson’s index of diversity were higher at untreated sites, and in 2018 diversity indices were higher at treated sites, with taxa richness increasing between years and higher evenness trending at treated sites. Our data suggest that treatment effects were potentially shrouded by natural variability of physicochemical variables, especially due to the varying hydroperiod observed over the three years of sampling. Additional work is needed to capture average conditions and separate confounding variables from treatment effects. This study provides an inventory of the current wetland insect community in the South March Highlands Conservation Forest landscape that offers a reference for ongoing mosquito management.

There is lack of evidence on which of the two highly recommended malaria prevention methods, insecticide treated bednets and indoor residual spraying, is more effective than the other. There is also limited peer reviewed literature that compares the characteristics of people who use the two malaria prevention methods. Based on the Health Belief Model, the research questions tested whether there is any relationship between the use of mosquito bednet or the use of indoor residual spraying and contracting malaria, and whether there is any relationship between sociodemographic and socioeconomic factors and the use of malaria prevention methods. Using a quantitative research design, secondary data from the 2011 Angola malaria indicator survey were analyzed. Chi-square for association, logistic regression, and multinomial logistic regression tests were used. There was no statistically significant association between the use of mosquito bednet and having malaria. However, the use of indoor residual spraying significantly reduced the likelihood of getting malaria. There was also a statistically significant association between place of residence, wealth index, level of education, and number of household members and using mosquito bednet and between wealth index and using indoor residual spraying. In conclusion, the malaria prevention programs should focus on indoor residual spraying. It is recommended that all households in southern Africa malaria prone areas should be regularly sprayed. The findings of this study contribute to positive social change in the sense that by using more effective malaria prevention method, individuals will be able to function normally on daily basis, save on expenses related to employment loses or treatment and care of the sick, as well as loss of life and improve own economic status.

Rift Valley Fever is a zoonotic, arthropod-borne disease that adversely affects ungulates and people. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus), is primarily transmitted through mosquito bites, yet can be transmitted by exposure to infectious aerosols. Presently, there are no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. Additionally, efficacy has been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, we characterize a hamster RVFV challenge model and use it to evaluate the activity of favipiravir against the highly pathogenic ZH501 strain of the virus. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days after infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater in hamsters challenged with RVFV when administered within 6 h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden in the brain observed in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

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.

Virtually all organisms exhibit circadian rhythms, this includes mosquitoes. Many aspects of their biology are under the control of their circadian clocks like their vision, olfaction, host-seeking, mating, oviposition, metabolism, locomotion, and more. However, how the circadian clock regulates mosquito-host interactions and adapts to specific environmental conditions remains largely unknown, despite its importance to vector disease control. Here, we relied on a multidisciplinary, integrative, and comparative approach to shed light on the mechanisms underlying mosquitoes adaptations to various temporal niches. We use CRISPR/Cas9 to knockout timeless in Aedes aegypti and show this mutation causes an increase in their free running periods under continuous darkness conditions. External factors can also influence a vector's activity pattern like climate, light pollution, as well as host preference and availability. To investigate the influence these factors have on activity patterns, we compare the activity patterns of multiple lab rear strains of Aedes albopictus as well as two field collected populations of Aedes japonicus. Our results suggest host availability and light pollution could cause the differences in activity profiles that we observed. With vision playing an important role in both circadian rhythms, host seeking, and threat detection, we compared a nocturnal mosquito (Aedes japonicus) and a diurnal mosquito (Aedes aegypti). We introduced a looming visual stimulus in an LED arena and found Aedes aegypti to be more responsive to the looming stimulus than Aedes japonicus. Finally, the underlying mechanisms that mediate this threat detection and escape behavior were examined, revealing a possible candidate for a giant fiber neuron in Aedes aegypti. Overall these results provide novel insights into mosquito biology that have the potential to be applied towards vector control methods. / Doctor of Philosophy / Most living things, including mosquitoes, follow a daily pattern called a circadian rhythm. This rhythm affects many parts of their lives including their vision, smell, and activity. Understanding how their circadian clock functions can help us control populations of mosquitoes and prevent the transmission of diseases they may carry. By studying different species and populations of mosquitoes in the lab and from the wild, we have found that factors like light pollution and host availability could affect when mosquitoes are active. In addition to these results we have also demonstrated that mosquitoes that are active during the day and mosquitoes that are active during the night will respond differently to a looming visual stimulus. We examined the brain of the mosquitoes that were most responsive to the looming stimulus and found a system of large neurons that could potentially be responsible for eliciting their escape behaviors. Overall these results provide new and important information about mosquito biology and can be applied to mosquito control.

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