3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Insecticide resistance charaterization, quantification, and transferal between life stages of the malaria vector Anopheles funestus giles (Diptera: Culicidae)(2015-04-22) Wood, Oliver RichardSouthern African pyrethroid resistant and insecticide susceptible laboratory colonies of the malaria vector Anopheles funestus were investigated to further understand the phenotypic expression of pyrethroid resistance and to establish at which life stage resistance was selected. Pyrethroid resistance levels of larvae and adults were assessed at the larval and adult life stages using WHO larval and CDC bottle bioassays. Subsequent resistance levels were then assessed following targeted selections at each life stage. Tests for an association between cuticle thickness and pyrethroid resistance were based on cuticle thickness measurements using scanning electron microscope imaging of prepared tissue sections. It is concluded that pyrethroid resistance in southern African An. funestus is only expressed in the adult life stage, and that selection for this phenotype can only be achieved by exposing adults. It also concluded that pyrethroid tolerant or resistant females are likely to have thicker cuticles than less tolerant or susceptible females.Item Insecticide resistance and Bionomics in laboratory reared and field caught Anopheles funestus Giles (Diptera: Culicidae)(2013-01-23) Spillings, Belinda LeaMalaria is transmitted by the mature, blood feeding portion of mosquito vector populations. Malaria vector control programs based on indoor residual spraying (IRS) of insecticides are designed to target resting adult Anopheles mosquitoes before or after they have blood fed. When a female mosquito acquires a blood meal, she could also ingest harmful xenobiotics that are present in the blood. During the resting period after feeding, many processes are initiated in order to assist in the digestion and assimilation of the blood. Ultimately, this enables the mosquito to absorb those amino acids needed for the biosynthesis of yolk proteins, which are essential for subsequent egg maturation. Since the regulation of xenobiotic (including insecticides) detoxification enzyme systems is likely to be altered in response to the ingestion of blood, this study aimed to investigate the effect of a blood meal on insecticide tolerance in insecticide resistant and susceptible southern African strains of the major malaria vector Anopheles funestus. Through the use of CDC bottle bioassays it was demonstrated that blood fed An. funestus carrying a pyrethroid resistant phenotype are even more tolerant of pyrethroid intoxication than their unfed counterparts. Using another major malaria vector, An. gambiae, microarray analysis revealed that a general increase in delta class glutathione-s-transferase (GST) expression occured in response to a blood meal. One gene, GSTD3, was over-expressed in both blood fed An. gambiae and An. funestus. Although this gene could not be validated with real time quantitative PCR, it serves as a viable target for future investigations. Since the pyrethroid resistant phenotype of southern African An. funestus has been linked to the over-expression of the duplicate copy gene CYP6P9, the expression levels of both copies of this gene were investigated. CYP6P9 and its copy, CYP6P13, showed a small but significant increase in expression in response to a blood meal. The increased expression of these major effect genes in response to blood feeding may be responsible for the increase in insecticide tolerance seen in the bottle bioassays. In an effort to repeat these experiments on wild caught An. funestus, field material was collected from Karonga in northern Malawi. Specimens were morphologically identified as members of the An. funestus group. However, attempts to molecularly identify them to species level failed. Through the use of ITS2 and D3 sequence analysis, cytogenetics and cross mating studies it was possible to conclude that these wild caught specimens were a new species. They have been provisionally named An. funestus-like.Item Genetic and biochemical characterization of the cytochrome P450, CYP6P9, associated with pyrethroid resistance in the African malaria vector Anopheles funestus(2012-01-19) Stradi, MelanieAnopheles funestus Giles is a major vector of malaria in Africa and pyrethroid resistance observed in this species has disrupted malaria control in southern Africa. Metabolic detoxification, based on the overproduction of cytochrome P450s, specifically CYP6P9 and CYP6P13, was identified as the principal resistance mechanism in both field and laboratory populations. This project aimed to characterize this resistance mechanism further, both on a molecular as well as a biochemical level. Biochemical analysis on total P450 activity levels revealed a 25.5-fold increase in the resistant strain compared to a pyrethroid susceptible strain. Analysis of the effect of pyrethroids on mRNA expression of three P450 genes showed that two of them (CYP6P9 and CYP6P13) as well as Cytochrome oxidase I (COI) was induced. HPLC analysis using a heterologously (recombinant) expressed CYP6P9 enzyme, showed that CYP6P9 was able to metabolize the pyrethroid permethrin and that it was catalytically efficient. Immunoblotting revealed no significant variation in CYP6P9 protein abundance between the different An. funestus colonies. Although an approximate molecular weight (≈Mr) of 58kDa was predicted for CYP6P9, two fragments were detected at ≈Mr 52,000 and ≈45,000. The smaller fragment was very likely a result of proteolytic degradation. Statistical analysis revealed there was no significant difference in CYP6P9 protein expression between strains or sexes. Although CYP6P9 mRNA is over-expressed it is important to assess the abundance of protein as well when elucidating whether a gene and its protein are important candidates in resistance. Differences in pyrethroid resistant or susceptible profiles of An. funestus colonies could be related to enzyme affinity for substrate and stability of CYP6P9 protein however; it is recommended that further studies need to be done before any conclusions can be drawn. CYP6P9 in An. funestus is a major candidate in conferring pyrethroid resistance and the pyrethroid resistant strain is able to metabolize the pyrethroid permethrin.Item The effects of pyrethroid resistance on transcription of metabolic enzymes in a major African Malaria vector, Anopheles funestus(2012-01-11) Christian, Riann NAnopheles funestus is a major vector of malaria in the southern African region. Insecticide resistance to pyrethroid and carbamate insecticides has been recorded in populations of this species in South Africa and Mozambique. This study aimed to determine the relationship between pyrethroid resistance and gene expression of two closely related genes, CYP6P9 and CYP6P13, by age and sex in a resistant strain An. funestus from southern Africa, FUMOZ-R. The insecticide susceptibility assays showed that percentage survival in both FUMOZ-R sexes significantly decreased as age increased. The mRNA expressions of CYP6P9 and CYP6P13 were higher in FUMOZ-R relative to the insecticide susceptible strain (FANG). The expression of permethrin resistance varies with age in An. funestus FUMOZ-R. The results indicate that other genes may also be involved in insecticide resistance. In addition to this, the expression profile of other metabolic genes involved in insecticide resistance was also investigated. A microarray based approach was used to identify genes differentially expressed in FUMOZ-R and FANG. As the full set of detoxification genes in An. funestus are unknown, this study investigated the utility of the An. gambiae detox chip to screen for differentially expressed detoxification genes in An. funestus. After optimization of the hybridisation conditions, over 90% of the probes showed a positive signal. Only three genes were significantly (P<0.001) differentially expressed in the females, CYP6P9, COI and CYP6M7. The same genes were also significantly differentially expressed in the adult males, together with an additional 21 transcripts. The third part of this study investigated the gene expression in the first instar, fourth instar and 3-day old adults in FUMOZ-R using the An. gambiae detox chip. The variation in metabolic enzyme gene transcription at the different developmental stages in An. funestus are not known. The identification of differentially transcribed genes at the different life stages provides some insight into the role and function of these genes. A large number of cytochrome P450s (monooxygenases), esterases, glutathione S-transferases (GSTs) and other additional genes were differentially expressed in all life stages. This study provided vital information regarding genes potentially involved in pyrethroid resistant and is the first to provide metabolic or detoxifying transcription gene information in An. funestus.