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Dengue is the most rapidly spreading vector borne disease. In Thailand, the number of dengue cases increased from 46,829 in 2006 to 76,059 by September 2008 (WHO, 2008). Insecticides play a vital role in controlling this disease but the success of control programmes is continually threatened by the emergence and spread of insecticide resistance in the vector population.

Aedes aegypti is the principle vector of dengue and yellow fever. This mosquito species is widely distributed throughout the tropics and international travel of goods and people have helped spread the mosquito and the diseases it transmits. Ae. aegypti has been recorded in the Cayman Islands four times in the past 45 years and each time efforts by the Mosquito Research and Control Unit have seemingly served to eliminate it. However, the current population of Ae. aegypti in Grand Cayman, believed to have been introduced in 2002, has proved refractory to control with current insecticide based methods. This study was conducted to assess the extent and potential causes of insecticide resistance in Ae. aegypti in Grand Cayman, investigate the likely origin of the 2002 introduction, and explore alternative methods of control. Ae. aegypti from Grand Cayman are highly resistant to DDT and pyrethroid insecticides but show only low level resistance to the organophosphate temephos. Glutathione-5- transferase, cytochrome P450 and esterase activities were elevated in adults of the Cayman Islands strains compared to a susceptible strain although use of the synergist piperonyl butoxide did not implicate increased insecticide detoxification as the major mechanism of resistance. Partial sequencing of the voltage gated sodium channel, the target site of DDT and pyrethroid insecticides, identified two single nucleotide polymorphisms, one within domain 11 56 region (V10161) and the other within domain III 56 (F1534C). A tetraplex PCR assay was designed to detect the latter mutation and its association with the insecticide resistance to permethrin and DDT was confirmed (Fisher's exact test P=O). Microsatellite analysis was employed to investigate the similarities between Cayman Islands Ae. aegypti and other populations collected from around the Caribbean. One hundred and fifty mosquitoes from three sites in the Cayman Islands and a total of 180 mosquitoes from four sites in neighbouring countries were genotyped at 12 loci. The allelic diversity suggests that the Cayman Islands population is well established, most likely receiving new introductions regularly. Fst analysis indicates closest comparative similarity to Jamaican populations of Ae. aegypti (Fst = 0.059-0.083). The high level of insecticide resistance prompted the search for alternative methods of Ae. aegypti control. The first field trial of a genetically modified sterile strain of mosquito (OX513A) was performed in collaboration with the company Oxitec Ltd. This strain carries a dominant lethal and mating with wild mosquitoes induces sterility. In the first field trial, this technology proved capable of significantly suppressing a wild Ae. aegypti population in a 16 Ha site in Grand Cayman (reduction in positive ovitraps in relation to control site, P<O.OOOl). The work carried out for this thesis has impacted upon decisions made by the department regarding insecticide choice for Ae. aegypti control. Knowledge of the mechanisms of resistance will further serve to assist in resistance management, and further studies of genetic control methods are planned.

Pyrethroids are currently the most widespread class of insecticide in public health use, although their future may be limited by resistance. The present study investigates the effects of these compounds on mosquitoes, other than mortality immediately after exposure. Longevity of both male and female An. gambiae was found to be significantly curtailed following sublethal exposure to the most commonly used pyrethroids, permethrin, deltamethrin and lambda-cyhalothrin. There was no direct effect observed on fecundity. Irritancy on direct contact with all 3 compounds was recorded, particularly with permethrin. Evening host-seeking flight activity was greatly reduced, and when females were treated with permethrin before release into a flight chamber, significantly fewer flew to a host to feed. Experiments investigating Plasmodium yoelii and P. falciparum development in the Asian vector An. stephensi revealed that sublethal exposure to pyrethroids inhibited development of oocysts in the midgut. Similar experiments using organochlorine, organophosphate and carbamate insecticides found no effect. Radio-labeled permethrin was traced inside the blood meal following exposure. However, none of the 3 pyrethroids were found to have a direct anti-malarial activity on cultured gametocytes. Likewise, permethrin did not affect exflagellation of P. yoelii. Time course experiments determined that for the reduction in oocysts there was a critical period of between 18 and 48 hours after the infective feed. Permethrin was not found to inhibit trypsin activity in assays, and the levels of trypsin in the midgut of blood fed treated and untreated females was not found to differ in a manner which could explain reduced infection rates. It is clear that sublethal exposure of mosquitoes can lead to a wide range of potentially important effects in terms of the impact of pyrethroids in malaria vector control.

In the South American and Southeast Asian forests, the Anopheles mosquito malaria vectors bite outdoors early in the evening, and rest outdoors. These behaviours preclude complete control by insecticides and bednets. Personal protection is necessary to protect from drug-resistant malaria. The thesis investigates the development of novel, plant-based mosquito control and personal protection: 1) discovery of new, culturally acceptable plants, 2) laboratory testing of those plants and 3) field testing of efficacious candidates as revealed by laboratory testing. Focus is upon on development of robust methodologies for the laboratory and field that carefully consider Anopheles behaviour to minimise bias. In Phase 1, an ethnobotanical survey in Yunnan, China, investigating knowledge, attitudes and practise about malaria and personal protection, several potentially insecticidal plants traditionally used against insects were identified. Knowledge of malaria was low, but everyone used personal protection at home, but not outdoors, to prevent nuisance biting. The expense of personal protection precluded use and the incorporation of plants into a low-cost insect repellent or mosquito coil would be acceptable for Yunnan. In Phase 2, a laboratory evaluation of neem (Azadirachta indica) as a larvicide, oviposition kairomone and bednet treatment, several assay methods were compared. WHO methodologies proved robust, and should be used as gold standards. When testing oviposition kairomones, choice tests should be run parallel with no-choice tests, as Anopheles mosquitoes oviposit in sub-optimal substrates in the absence of choice. A novel laboratory assay representing a normal exposure of bednet treatment to host-seeking mosquitoes was developed. In Phase 3, a field evaluation of plant based repellents in Bolivia, focus groups and novel repellents were investigated. Volatilisation of repellents proved unsuitable for use in open housing. 20%, of mosquitoes, are diverted from deet repellent-protected to unprotected individuals. The implications for repellent-testing methodology, and repellents as a means of disease control, are discussed.

Malaria, the leading cause of death due to vector-borne disease. is responsible for around 250 million cases and 1 million deaths annually, mostly in sub-Saharan Africa where Anopheles gambiae is the major vector. Novel approaches to malaria control include theoretical use of transgenic mosquitoes to suppress or replace wild type populations. Generation of mosquitoes carrying either a conditional lethal gene (RIDL), or one encoding a molecule able to kill malaria parasites, is a key component of this strategy. Detailed genetic and physiological knowledge of transgenic mosquitoes is important for epidemiological models to accurately determine the outcome of putative field releases. This includes testing the potential for post-integration remobilisation of transgenes in the host genome and testing the reproductive fitness of transgenic mosquitoes in comparison to wild type. Complex plasmids, designed to create both RIDL strains and strains within which piggyBac remobilisation could be tested, were injected into the Keele strain of An. gambiae. Ultimately, although microinjection techniques were optimised and efficiencies improved over time, generation of such transgenic strains was unsuccessful. A previously established transgenic strain (EVida3), expressing the Vida3 peptide which is active against ookinete stages of Plasmodium, was subsequently investigated with respect to multiple feeding and relative fitness. Multiple feeding experiments indicate that complex interactions take place between Vida3 and the natural immune system when mosquitoes are infected with the murine malaria P. yoelii nigeriensis. Vida3 production appears to impact on parasite intensity and mosquito fecundity in both positive and negative ways dependent on factors such as gonotrophic cycle and parasite-stage. Analysis of three life-table fitness parameters, comparing EVida3 hemizygotes to the KIL wild type laboratory strain, indicate no fitness cost associated with transgene presence or expression. Analysis of a further two parameters indicates a possible fitness cost associated with inbreeding rather than transgenesis alone.