A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science Johannesburg, 2014 / Southern 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.

Anopheles 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.

A thesis submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2014 / Malaria is the most serious tropical infectious disease in humans, caused by parasites of the Plasmodium genus and transmitted by anopheline mosquitoes. The interaction between the parasites and vectors has become a focus for malaria research as it may present an alternative disease control method by enhancing anti-plasmodial factors within the mosquito to impede parasite development and transmission. Anopheles gambiae is the best studied African malaria vector and is often used with the murine malaria parasite, Plasmodium berghei, for investigating parasite-vector interactions in the laboratory setting. Anopheles funestus has not been studied and its interactions with Plasmodium were unknown, until now. Although the Vector Control Reference Laboratory routinely maintains An. funestus and a number of An. gambiae colonies, none have been infected with Plasmodium since their establishment. This study aimed to use P. berghei to determine the vectorial capacity of these colonies and to examine the involvement of the 2La paracentric chromosomal inversion and antimicrobial peptides during Plasmodium infection in An. gambiae and An. funestus, respectively. Most of the An. gambiae complex colonies were susceptible to P. berghei, but the range of feeding and infection rates varied considerably. The infection rates for some of the older colonies were lower than previously documented. Anopheles funestus colonies were all viable vectors and there was an inverse correlation between the insecticide resistance profile and parasite susceptibility. Increased detoxification enzyme activities may have been contributing to a greater degree of parasite elimination. In An. gambiae, molecular karyotyping of the 2La inversion using PCR was validated against traditional cytogenetic techniques. The PCR was shown to be a reliable substitute for identifying the inversion. Using molecular karyotyping on 2La polymorphic colonies infected with P. berghei, it was found that infected females were more likely to carry the 2La inversion, indicating possible correlation between the inversion and susceptibility to parasites. In An. funestus, the expression of antimicrobial pepetide genes during P. berghei infection was examined using real-time PCR. Although all three genes showed increased activity at certain points of the infection, none displayed significant anti-plasmodial properties. However, in the less parasite susceptible strain, expression of two genes was higher towards the end of the infection, which was not observed in the other strains. It is possible that the co-expression of both peptides has led to a decrease in parasite load in late infection, but given the multi-factorial nature of the parasite-vector interaction, further investigation is required.

The primary vector of malaria in the eastern United States, Anopheles quadrimaculatus (Say), was recently discovered to be a complex of five different cryptic species: A - An. quadrimaculatus, B - An. smaragdinus, C1 - An. diluvialis, C2 - An. Inundatus, D - An. maverlius (Reinert et al. 1997). In this research project, the goals were to determine which species were found in Texas, establish overall distribution patterns of those species, and observe the dates in which each specimens were collected so that any seasonal changes in species could be observed. Both An. quadrimaculatus (A) and An. smaragdinus (B) were identified from collections made throughout Texas from September 2002 through January 2005. Anopheles smaragdinus only made up 3% of the total specimens collected and neither An. inundatus nor An. maverlius were collected in Texas, even though they have both been collected in neighboring Parishes in Louisiana. Anopheles. quadrimaculatus' habitat and geographic range was found to be more extensive than An. smaragdinus. While An. smaragdinus was found only in the easternhalf of Texas with no collection south of Fort Bend County, An. quadrimaculatus was found throughout the eastern half of Texas, many of the southern Golf coast counties, and a few counties in far west Texas. The most common land cover where An. quadrimaculatus specimens were collected was on pasture/hay fields. This is very different from An. smaragdinus specimens in that pasture/hay was one of the least common land covers and the dominant land cover was woody wetlands. Overall, An. smaragdinus was usually associated with land covers that could provide shelter, while An. quadrimaculatus could be found among habitat that was more open and urban. There was no observed change in the species composition over time in this study. In fact, when An. smaragdinus was collected, An. quadrimaculatus was usually collected at the same time. Both An. quadrimaculatus and An. smaragdinus were collected throughout late spring, summer and early fall. Of course, the collection times of these species could have been an artifact of when most of the collectors were looking for An. quadrimaculatus (Say) specimens.

The primary vector of malaria in the eastern United States, Anopheles quadrimaculatus (Say), was recently discovered to be a complex of five different cryptic species: A - An. quadrimaculatus, B - An. smaragdinus, C1 - An. diluvialis, C2 - An. Inundatus, D - An. maverlius (Reinert et al. 1997). In this research project, the goals were to determine which species were found in Texas, establish overall distribution patterns of those species, and observe the dates in which each specimens were collected so that any seasonal changes in species could be observed. Both An. quadrimaculatus (A) and An. smaragdinus (B) were identified from collections made throughout Texas from September 2002 through January 2005. Anopheles smaragdinus only made up 3% of the total specimens collected and neither An. inundatus nor An. maverlius were collected in Texas, even though they have both been collected in neighboring Parishes in Louisiana. Anopheles. quadrimaculatus' habitat and geographic range was found to be more extensive than An. smaragdinus. While An. smaragdinus was found only in the easternhalf of Texas with no collection south of Fort Bend County, An. quadrimaculatus was found throughout the eastern half of Texas, many of the southern Golf coast counties, and a few counties in far west Texas. The most common land cover where An. quadrimaculatus specimens were collected was on pasture/hay fields. This is very different from An. smaragdinus specimens in that pasture/hay was one of the least common land covers and the dominant land cover was woody wetlands. Overall, An. smaragdinus was usually associated with land covers that could provide shelter, while An. quadrimaculatus could be found among habitat that was more open and urban. There was no observed change in the species composition over time in this study. In fact, when An. smaragdinus was collected, An. quadrimaculatus was usually collected at the same time. Both An. quadrimaculatus and An. smaragdinus were collected throughout late spring, summer and early fall. Of course, the collection times of these species could have been an artifact of when most of the collectors were looking for An. quadrimaculatus (Say) specimens.

Thèse de doctorat : Sciences de la Vie et de la Santé. Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg : 2009. / Titre provenant de l'écran-titre. Bibliogr. 12 p.

Thesis (Ph. D.)--Rijksuniversiteit te Leiden, 1935. / Folded leaf with six theses entitled "Stellingen" inserted. Includes bibliographical references (p. [246]-256).

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 1982.

Thesis (Ph. D.)--Rijksuniversiteit te Leiden, 1935. / Folded leaf with six theses entitled "Stellingen" inserted. Includes bibliographical references (p. [246]-256).

Thesis (doctoral)--Rijksuniversiteit te Leiden, 1970.