No description available.

Bacillus thuringiensis subsp. israelensis (B.t.i.) produces a plasmid encoded parasporal crystalline protein which is larvacidal to mosquitoes carrying parasites for malaria and other infectious diseases. The purpose of this study was to construct a library of random fragments from the nine plasmids of wild type B.t.i. strain 402. The library was to be utilized in order to clone a 135kDa mosquitocidal polypeptide carried on a 108 kb B.t.i. plasmid.The library construction involved isolation of plasmid DNA by equilibrium density centrifugation, generation of random fragments of the nine plasmids by a partial Sau3A restriction digest, and ligation of these fragments into XbaI-BamHI restricted Lambda GEM-11 vector. Escherichia coli strain LE392 was infected by the packaged recombinant lambda and over 1000plaques were pooled to comprise the library. In order to verify construction of the library, both plaque screens of the library and Southern Analysis of restricted clones subjected to agarose gel electrophoresis were performed with labeled probes. The labeled probes were included: 1) radioactive end-labeled oligonucleotides constructed from published sequences of the B.t.i. 135 kDa toxic protein, 2) radioactive end-labeled random fragments from all nine plasmids of B.t.i., 3) radiolabeled entire plasmids of all nine plasmids of B.t.i., and 4) dioxigenin-labeled oligonucleotides. No homology between the lambda library digested DNA and the B.t.i. plasmid was observed. The results suggested that no lambda library of B.t.i. was constructed and, therefore, a lambda clone encoding the 135 kDa mosquitocidal polypeptide was not isolated. / Department of Biology

Mosquito-borne pathogens are the cause of important, often deadly diseases worldwide; and, use of repellents is one of the components of mosquito control programs. The most effective mosquito repellent is N,N-dimethyl-m-toluamide, known as DEET. Speculation on the adverse effects of DEET have been on the rise since the 1980s, and manufacturers are searching for a safe and long lasting insect repellent that may be used on the skin as well as possess spatial applications. Olfactometers used in the bioassays for evaluating repellents usually do not measure repellency, but rather measure non-attraction. Therefore, I designed a novel olfactometer that divides host-seeking behavior of mosquitoes into attraction and repellency at close range. I compared my olfactometer with another similar olfactometer and found no difference when attraction was measured. However, the new olfactometer assesses true repellency. I also added a new term to the terminology of host-seeking behavior, that is 'inhibition'. Inhibition occurs in combination with at least two chemicals and causes failure of mosquitoes to respond to a previously attractant stimulus. I evaluated 80 inert and active ingredients of commercially available products on eleven volunteers and determined that four were true repellents. I unexpectedly discovered that DEET did not attract nor repel the mosquitoes when applied on skin. I conducted a series of experiments to elucidate the behavioral mode of action of DEET. Lactic acid was used as a control attractant. I demonstrated that DEET attracted mosquitoes in the absence of a host. When DEET was combined with lactic acid, it neither repelled nor attracted mosquitoes. I concluded that DEET works in combination with lactic acid on the skin by inhibiting lactic acid, rather than being a true repellent. In this study, I developed a novel device measuring attraction, repellency and inhibition. I contributed to the mode of action of DEET by proving that DEET is not a repellent but an inhibitor, and I identified five true repellents that are worth consideration. / Graduation date: 1998

No description available.

The present product development project was aimed at studying the synergism and/or antagonism amongst various known insect repellent actives with the view to formulating a multi-active repellent product with improved properties when compared to current single-active commercial products. Advanced statistical methods were used to identify synergism between individual active substances and to define a formulation as close as possible to the “ideal” formulation. Several mosquito repellent samples were prepared and sent to the South African Bureau of Standards (SABS) in Pretoria to test for their efficiency in repelling mosquitoes. From the results of the repellency tests of the various active combinations, three actives were identified that showed promising signs of synergism. These actives were then studied in further detail to determine their optimum combination. In addition, it was shown that when using a natural flavourant as promoter and incorporating a slow-release agent into formulations for aerosols and lotions, a product is obtained that gives comparable levels of efficiency to current commercial products, but at much reduced levels of active loading. Accelerated stability tests performed on the final combination of the three actives used in the final formulation showed no adverse reactions over a three-week study. These tests shall be repeated once the final application form (lotion, aerosol, etc) and product packing have been decided.

Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus. / Department of Biology

Thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirement for the degree of Doctor of Philosophy 2019 School of Public Health University of the Witwatersrand, South Africa / Background: Spatial targeting of interventions is increasingly recognized as essential for malaria control, particularly in areas aiming for elimination. The associations between house characteristics and malaria transmission is known, but gaps remain on whether transmission is also influenced by factors such as distances between households or the degree to which houses are clustered. It is also important to identify new interventions that can be implemented by targeting critical points in the biology of major malaria vectors and common characteristics of human settlements. Such efforts will be essential to complement current major tools. Aims: This study had the following aims; (i) understand how households densities and their distances influence malaria transmission the study area , (ii) identify housing-related factors affecting residual transmission risks in the area (iii) assess opinions of residents on how house characteristics, settlement patterns and other environmental factors influencing malaria transmission, and (iv) identify effective complementary approaches that could be used to improve malaria control in these residual transmission area with high coverage of basic interventions such as long lasting treated nets. Methodology: A mixed methods approach was used, which included: (i) indoor and outdoor mosquito collections from fixed as well as randomly selected households over 12 months, (ii) characterization of physical and microclimatic conditions of the houses and surrounding environmental variables, (iii) assessing effects of spatial clustering of households on malaria transmission risks, and (iv) assessing community concerns regarding household and environmental factors influencing transmission. During these studies, swarms of Anopheles mosquitoes were discovered for the first time in this area. Therefore, additional quantitative and qualitative observations were made to characterize the swarms and assess how they could be targeted to complement malaria control efforts. Key findings: Despite the expansive use of LLINs in the area, factors associated with housing characteristics and settlement patterns as well as people’s perceptions contribute to persistent malaria transmission, and will need to be addressed for eventual elimination to be reached. The main findings of this study were as follows: (i) household occupancy influenced indoor mosquito density (ii) high house densities increased Anopheles biting risk but mosquito density declined as distances between houses increased beyond 50m; (iii) abundance of the vector Anopheles arabiensis peaked during high rainfall months (February-May), but An. funestus densities remained stable into the dry season (May-August); (iv) there were higher indoor densities of An. arabiensis and An. funestus in houses with mud walls compared to plastered or brick walls, open eaves compared to closed eaves and unscreened windows compared to screened windows; (v) most respondents were aware of associations between their house characteristics and malaria risks but carrying out improvements was constrained by financial costs and other household priorities; (vi) in all our surveys, An. funestus mosquitoes contributed approximately 85% of ongoing malaria transmission in the areas, even when occurring in far smaller densities than An. arabiensis; and (vii) with regards to complementary interventions, small scale studies demonstrated that mating swarms of both An. arabiensis and An. funestus could be readily identified and characterized (in this case by volunteer community members), and that targeting the swarms using aerosol spraying could possibly reduce overall biting risk in the communities. Conclusions: This study yielded evidence that malaria transmission risks are significantly lower when the distances between houses are high. Variation in malaria transmission risk within a village might be important for planning and implementing spatially targeted interventions. With limited resources, malaria control efforts must be prioritized in locations with significantly higher risk in order to obtain maximum benefit. Thus, the distances between houses should be considered when planning malaria vector control strategies. Furthermore, the overall clustering of indoor densities of major disease-transmitting mosquitoes is significantly associated with household occupancy and its spatial distribution within the villages. Further research is needed to assess the potential of using this information for predicting, identifying and targeting the most intense foci of mosquito-borne disease transmission based on household occupancy. Understanding the factors influencing malaria transmission at a small scale is crucial for planning malaria control interventions. Malaria transmission heterogeneity does not only exist on a large scale, but also at small scales, which may be influenced by small variations of environmental features, such as seasonality. These variations should be considered when improving malaria control interventions. While An. funestus showed variation in density during the year, numbers of mosquitoes remained high throughout the dry season, whereas An. arabiensis showed a definite peak in the rainy season with a subsequent drop during the dry season. Further studies are required to investigate the survival strategies of major malaria vectors during the dry season. Understanding this particular feature of An. funestus may lead to the design of new control strategies or improve existing interventions so as to reduce the malaria burden in such rural areas. This study also documented high mosquito densities in mud-walled houses compared to houses with brick or plastered walls. These findings further indicate the necessity of considering house improvement as a malaria control strategy. While the community members had fairly high awareness that improved houses were associated with reduced mosquito nuisance, many families in rural areas are living in poorly constructed houses. It is therefore necessary to consider inter-sectoral collaborations to integrate housing into health policies in Tanzania. Further studies are also needed to provide epidemiological evidence on how housing design affects malaria transmission. Furthermore, community-based house improvement programs should be promoted, including simple, scalable and affordable house improvements for the populations living in high malaria endemic areas in rural communities. Generally, this study has generated information which sets the basis for further studies on the relationship between the exact house location and malaria transmission risk in rural malaria endemic countries. / MT 2020

The Santa Cruz valley in Arizona is a rapidly urbanizing area. Complaints by residents of the area about pest mosquitoes prompted the investigation of mosquito breeding sources and a search for management techniques that would reduce mosquito populations. Many types of mosquito breeding sites were found in the area, and eight species of mosquitoes were identified. The greatest source of mosquitoes was a 2400 hectare irrigated pecan orchard. The most numerous and annoying mosquitoes were Aedes vexans (Meigen) and Psorophora columbiae (Dyar and Knab). The orchard is irrigated ca. every two weeks from April through October by flooding level areas, called borders, between rows of trees. Mosquitoes hatched with every irrigation studied, from April through September, but reached annoying numbers from late April through mid-September. A. vexans was the dominant species in early spring and P. columbiae was dominant in summer. Highest populations were reached coincident with the summer rainy season in July and August. Tests were performed to determine the efficacy of Bacillus thuringiensis israelensis (B.t.i.) as an additive to irrigation water for control of mosquito larvae. The larvicidal material was effective at all concentrations from .586 to 2.344 l/ha and with all techniques used. The most cost-effective application technique was to use fertilizer tanks to drip a mixture of B.t.i. into irrigation water in ditches before the water ran into fields. The general rate of three parts larvicide per million parts irrigation water was found to be effective, especially when supplemented with a spray of one part larvicide to 64 parts water applied to the ends of borders two to three days following irrigation. An increase in larvicide concentration was found to be necessary during the peak of mosquito season. A successful management program was developed and applied for 1 year, during which no mosquito complaints were made by citizens, the population of mosquitoes in the orchard was reduced to a point where farm laborers were no longer annoyed, and farm managers were satisfied that the program was cost effective.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Science, 1998. / This study was initiated in order to determine the identities and distribution patterns of the anopheline fauna, more especially the malaria vectors, in regions of Botswana prone to malaria epidemics. Field samples collected from Shakawe, Maun and Kasane over three consecutive years were subjected to morphological, cytogenetic, isoenzyme and PCR analyses. The results established that Anopheles arabiensis and Anopheles funestus were the predominant vector species.

Various strains of Bacillus thuringiensis Mt.) produce crystalline endotoxins specific for larvae of different insect classes. Two strains, B.t. subspp. israelensis and kurstaki produce similar 130 kDa toxins encoded by the CryIVB gene (toxic to Diptera) and the CryIA gene (toxic to Lepidoptera), respectively. The N-terminal region of the CryIVB gene was cloned into the Escherichia coli expression vector pKX223-3. A mosquitocidal transformant was obtained as determined by mosquito bioassays. The gene fragment, if stable, can be cloned into cyanobacteria to achieve biological control of mosquito-borne diseases. A second goal was to identify the binding specificity domain of the CryIVB gene which encodes the portion of the protein toxin that binds the insect midgut causing cell lysis and death. Two potential insect binding specificity domains identified by computer analyses were switched with a known binding specificity region of the CryIA gene. The polymerase chain reaction was utilized to obtain gene fragments of the CryIVB gene which replaced the CryIA gene binding specificity domain. The resulting recombinant clones carrying the CryIA gene containing the .000nd proposed insect binding specificity domain of the CryIVB gene were fotsd to be mosquitocidal. / Department of Biology