Thesis submitted to the Faculty of Health Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017 / Malaria remains endemic in the north-eastern areas of KwaZulu-Natal (KZN), Mpumalanga and Limpopo provinces of South Africa (SA). Anopheles arabiensis is now implicated as the main malaria vector. This vector is not completely amenable to current vector control strategies which target indoor biting and resting mosquitoes. SA is moving towards malaria elimination and there is a need for additional vector control interventions to complement existing tools. The sterile insect technique (SIT) targeting An. arabiensis was selected as a potential intervention. In a mosquito SIT programme, only sterile males should be released because females are potential disease vectors. In order to achieve male releases only, a reliable sex separation strategy is needed. Additionally, it is imperative to gather entomological baseline information on the population density, species composition, and vectorial capacity of the targeted wild population. The aim of this study was to evaluate the use of a local genetic sexing strain for SIT and to determine the population dynamics of wild An. arabiensis in northern KZN. The following objectives were initiated in this study: development of a local genetic sexing strain (GSS), evaluation of the life history and reproductive effects of irradiation on An. arabiensis, and weekly mosquito surveillance was conducted over a period of 24 months. A local GSS named GMK was established by introgressing a local wild-type population of An. arabiensis with an available GSS strain. The strain exhibited the following attributes: low egg hatch rates, fast developmental time, long adult survival and a high mating competitiveness. Dieldrin treatment of GMK eggs/larvae mainly produced males but this result remains controversial. The irradiation dose of 70 Gy induced male sterility without compromising their mating competitiveness and impacted negatively on female fitness, but not vectorial capacity. The perennial presence of An. arabiensis, the dominant anopheline species in Mamfene, was confirmed. Its population density fluctuated with season reaching a peak in summer. Clay pots were more productive than the other collection methods, collecting 16.3 mosquitoes per trap. This study recorded for the first time wild caught An. arabiensis and An. vaneedeni infected with P. falciparum in SA. An arabiensis sporozoite infection rates were 0.7% (2014) and 0.5% (2015). Anopheles vaneedeni has never been implicated as a malaria vector in nature. However, an infection rate of 1.96% was recorded (2014-2015), which implicate this species as a potential malaria vector. These results highlight the importance of intensive mosquito surveillance to establish malaria vectors responsible for low level/residual malaria transmission. The data generated provides important baseline vector surveillance information and is valuable to stakeholders and researchers to make informed decisions regarding the use of SIT against vector mosquitoes in SA. / MT 2018

Malaria is the leading cause of mortality and morbidity in Sudan. The annual malaria cases and deaths are estimated at 7.5 million and 35, 000 respectively. One of the possible factors that have led to this situation is the development of insecticide resistance in the main malaria vector in Sudan, Anopheles arabiensis. This study therefore, was initiated to identify the malaria vectors in Gezira and Sennar states of central Sudan, determine their susceptibility levels to the different classes of insecticides used for malaria vector control, identify mechanisms of resistance, and determine the sporozoite infection rate and the blood meal sources in these populations. The polymerase chain reaction (PCR) for species identification revealed that An. arabiensis was the only member of the An. gambiae complex present in the study area. The blood meal analysis using ELISA showed high anthropophily with 89.2% feeding on humans. The overall sporozoite infection rate was 2.3 %. WHO susceptibility tests showed complete susceptibility of An. arabiensis to bendiocarb (100% mortality) and multiple resistance to permethrin (54-78%), DDT (55-66%) and malathion (76-78%). The kdr mutation analysis revealed the presence of the West African kdr allele with the majority of specimens being heterozygous (RS). The kdr in DDT/permethrin susceptible specimens were: 15% homozygous for the kdr mutation (RR), 64.2% heterozygous (RS) and 20.8% homozygous for the susceptible allele (SS). Amongst the DDT/permethrin resistant specimens, 13% were SS, 48.7% RS and 38.3% RR. The apparent lack of correlation between kdr and resistant phenotype strongly suggests that other resistance mechanisms are playing a role.

Malaria continues to be a significant cause of morbidity and mortality in the developing world with Africa being the most affected. Malaria vector control with chemical insecticides is the primary intervention to curb transmission. However, the success of chemical based interventions is threatened by the escalating development of insecticide resistance in the major anopheline mosquito vectors.

0009014A Msc thesis Science Animal plant and environmental sciences / Anopheles arabiensis is considered an important vector of human malaria in the southern African region where the disease is the major cause of morbidity and mortality. Gene flow plays an important role in malaria control with the spread of insecticide resistance. The main objectives of this study were to (i) measure the genetic variability within and between five populations (Botswana, Namibia, South Africa, Swaziland and Zimbabwe) of wild An. arabiensis and (ii) estimate the level of gene flow between natural populations across the Southern-limits of An. arabiensis. A total of 1225 An. arabiensis specimens were identified out of 1300 mosquitoes collected from 2000-2003 with the sample sizes ranging from 180-292 per country. Variation at four microsatellite markers was investigated on non-denaturing polyacrylamide gels. The results showed fewer variations between populations (2.96%) than within populations (82.60%) suggesting considerable homogeneity. However, estimates of gene flow (Nm) calculated from mean FST and RST statistics were relatively low, 1.14 and 1.19 respectively, suggesting somewhat restricted gene flow between populations. The occurrence of gene flow within subpopulations of An. arabiensis in Zimbabwe but not in South Africa is interesting with regard to the spread of insecticide resistance in Zimbabwe. The results presented here are obviously subject to the limitations inherent in manual, silver staining method of analysing microsatellite DNA markers. It is possible that a different set of results would be obtained if an Automated Sequencing Analyzer were used. ii

Opportunistic feeding behaviour and partial exophily make An. arabiensis much more difficult to control by indoor residual spraying than any other vector in the Afro-tropical region. The persistent malaria outbreaks in Zimbabwe despite decades of indoor house spraying prompted this investigation into the role of An. arabiensis in malaria transmission and assessment of the possible impact of this control measure. The study was conducted in the malaria endemic districts of Binga and Gokwe. An. gambiae complex mosquitoes were collected from artificial outdoor resting sites, and from human dwellings by i) daytime hut searches, ii) pyrethrum spray catches and iii) exit window traps. Mosquito components were processed to enable: i) the distinction of An. arabiensis from An. quadriannulatus and An. merus on the basis of the pale band at the junction of the hind leg 3/4 tarsomeres; ii) species identification and scoring of inversion polymorphism on the basis of the X chromosome and autosomes respectively; iii) the determination of blood meal sources using the Ouchterlony precipitin test; and iv) identification of An. gambiae s.l. using polymerase chain reaction (PCR) and enzyme electrophoresis techniques. Entomological assessment of residual spraying included determining: the vector resting densities indoors and outdoors, bioassay and insecticides susceptibility tests. Data were also collected on hut profiles, knowledge-attitudes-practices surveys, and household malaria prevalence . surveys. An. arabiensis and An. quadriannulatus were found in sympatry in Binga and Gokwe, and in addition, An. merus was found in Gokwe. Most species identifications were made using PCR; which was found to have 7.5% and 41.6% levels of error for An. arabiensis and An. quadriannulatus respectively, using the cytogenetic technique as benchmark. The pale band technique yielded > 80% correct identification for An. arabiensis but the extent of overlap in the pale band lengths between An. arabiensis and An. quadriannulatus renders the method unsuitable for distinguishing these two species. Inversions 2Rb and 3Ra were found floating in An. arabiensis, with 60% frequency in the former. The Wright's F statistic value of -0.0416 indicated an excess of heterozygotes, and a state of panmixis in the vector population. No significant differences were observed between 2Rb karyotypes in host choice. Human blood indices among indoor (0.82), exit trap (0.98) and outdoor resting (0.30) specimens suggested exophilic behaviour. This was corroborated by the high fed:gravid ratios of 6.8: 1 and 11.6: 1 in sprayed and non-sprayed dwellings respectively. This was worsened by a high feeder-survivor index (FSI) of 93 % among exit trap specimens. The susceptibility to deltamethrin coupled with residual efficacy nine weeks post-spray indicated the suitability of the insecticide. Rural dwellings were suitably built for spraying but had no mosquito proofing. Personal protective measures are hardly known; sleeping outdoors occurs in Siabuwa. While An. arabiensis bites humans indoors the partial exophily it exhibits is a threat to indoor residual insecticide spraying. An integrated malaria control approach is recommended. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Les femelles moustiques peuvent être vectrices de nombreux agents infectieux (virus, protozoaires, helminthes) pour l'Homme, qui peuvent être la cause de maladies graves comme le paludisme et la dengue. Ces maladies menacent respectivement 50 et 40% de la population mondiale ; le paludisme étant responsable de près d'un million de décès par an. Les méthodes de lutte anti-vectorielle destinées à limiter les populations vectrices et stopper la transmission de maladies, se heurtent au développement incessant de résistances de la part des moustiques et des agents infectieux vis-à-vis des traitements employés. Bien que certaines régions du monde aient réussi à stopper efficacement la transmission de certaines de ces maladies, une grande partie des régions tropicales reste menacée. De plus l'expansion rapide de certaines espèces vectrices, telles qu'Aedes albopictus, accroît les risques sanitaires dans de nouvelles régions du globe. La technique de l'insecte stérile (TIS), qui a permis l'éradication ou la suppression des populations de nombreux insectes nuisibles aux cultures et à l'Homme, représente un moyen de lutte prometteur contre les moustiques. Cette technique s'appuie sur le lâcher en masse de mâles stérilisés par rayonnements ionisants qui, en transférant un sperme stérile aux femelles sauvages, vont permettre une diminution progressive de la population cible. Suite à l'épidémie de chikungunya à l'île de la Réunion en 2005 et face aux menaces permanentes de recrudescence de la dengue et du paludisme, les services de lutte anti-vectorielle réunionnais mettent en place d'importants moyens de lutte contre les populations de moustiques concernées. Toutefois, ces mesures ne permettant pas une diminution durable des densités de vecteurs, une étude de faisabilité est en cours quant à l'utilisation de la TIS pour diminuer et contrôler les populations d’Aedes albopictus, vecteur de la dengue et du chikungunya, et d’Anopheles arabiensis, vecteur du paludisme.Ce travail de thèse s'inscrit dans le cadre du projet TIS Réunion, dans le but d'étudier la biologie et le comportement des souches destinées aux lâchers de mâles stériles. Dans un premier temps, cette étude s'intéresse à la comparaison entre les souches d'élevage d’Anopheles arabiensis et les souches sauvages, ainsi qu'aux modalités de stérilisation des mâles de la souche à sexage génétique. Une seconde partie est consacrée à l'étude de l'effet de l'irradiation sur les mâles d’Aedes albopictus, en étudiant plus particulièrement leur stratégie de reproduction, leur capacité d'insémination en laboratoire, ainsi que leur compétitivité sexuelle et longévité face aux mâles sauvages en conditions semi-contrôlées. / Mosquito females are potential vectors of numerous pathogens (viruses, protozoa, helminths), which can cause serious diseases such as malaria and dengue in humans. These two infectious diseases are threatening 50 and 40% of the world population respectively. Malaria is responsible for nearly one million deaths per year, and is considered by many experts as the most important insect-transmitted disease. Antivectorial control methods, intended to limit the vector populations and to stop the disease transmission have to face many challenges such as the development of mosquitoes' and pathogens' resistance to the treatments employed to control them. Although various regions of the world have succeeded in efficiently stopping the transmission of some diseases, most of the tropical regions remain under threat. In addition, the rapid expansion of some vector species, such as Aedes albopictus, increases the risks in previously safe areas of the world. The sterile insect technique (SIT) has allowed the eradication or suppression of various insect pest populations threatening crops, animal, and human health, and could offer a promising control tool against mosquitoes. The classical SIT relies on the mass releases of males sterilized by ionizing radiation;they transfer sterile sperm to wild females, which results in a progressive reduction of the target population.Following the chikungunya outbreak in Reunion Island in 2005 and considering the constant threat of arecrudescence of dengue and malaria, the anti-vectorial services in Reunion Island are deploying important means to control the relevant mosquito populations. However, these measures do not confer a permanent, or long-lasting reduction of vector densities. A feasibility study is ongoing, evaluating the use of the SIT to diminish and control the populations of Ae. albopictus, a vector of dengue and chikungunya, and Anopheles arabiensis, a vector of malaria. This PhD work was developed in the context of the SIT Reunion project, with the aim of studying the biology and the behaviour of some strains intended for the sterile male releases. Firstly, this study endeavours to compare colonized and wild strains of An. arabiensis, and to determine the sterilisation procedures of the genetic sexing strain males. The second part of this work studies the effect of irradiation on male Ae. albopictus, and most notably their reproductive strategy, the insemination capacity in laboratory, and finally their sexual competitiveness and longevity against wild males under semi-field conditions.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although malaria remains a major public health concern, especially in sub-Saharan Africa, little information exists on the physiological tolerances of malaria vectors. Here, I aimed to provide a comprehensive set of physiological tolerances for Anopheles arabiensis and An. funestus, by investigating thermal tolerance traits of adults, larvae and pupae; desiccation resistance of adults and development rate-temperature relationships for both vectors. Critical thermal limit (CT) and desiccation data showed significant effects of increasing adult age on reducing tolerance to temperature or dry conditions. Females of both species were more tolerant of high or low temperatures in CT experiments and were more desiccation tolerant than males in desiccation trials. Anopheles funestus was more desiccation tolerant than An. arabiensis, despite the common misconception that An. arabiensis is the more arid-adapted of the two species. Comparisons between thermal tolerance traits of adult laboratory and wild strain progeny of both species indicated a high degree of similarity between critical thermal limits in wild and laboratory strains, suggesting that the use of laboratory populations of both mosquito strains can provide an accurate estimate of wild population responses to thermal change. Lethal temperature estimates for both vectors indicated a higher tolerance to high temperature in An. arabiensis larvae and pupae when compared with An. funestus, and a greater tolerance of high or low temperatures in adult females when compared with adult males. Species differences between the vectors were further highlighted in development rate-temperature experiments. Under fluctuating and constant temperatures, An. arabiensis developed significantly faster than An. funestus and had higher survival to the adult stage. Under fluctuating temperatures, An. arabiensis developed faster or no different to constant temperatures, while survival under fluctuating temperatures was also comparable to constant temperature estimates. This faster development rate of this species is likely a consequence of the puddle-breeding nature of An. arabiensis and the need to develop to adulthood before evaporation of breeding sites. Anopheles funestus on the other hand, showed reduced survival and development under fluctuating temperatures when compared with constant temperatures, probably as a result of the more thermally stable breeding sites usually used by this species. Distribution data of these species, combined with developmental parameters in a process-based distribution model, suggests that both species will show range changes in response to climate change. Areas where these species were previously only present on a seasonal basis might become more suitable for vector population establishment and persistence, while areas on the northern margins of current distributions will become less favourable, leading to an overall southerly shift in habitat suitability for both species. Increases in temperature and changes in rainfall patterns as predicted to occur with climate change are likely to impact the distribution of both malaria vectors. Combining the physiological tolerance data collected in this thesis in a future, planned mechanistic distribution model, will provide an accurate indication of potential range shifts of these vectors and hence, provide an indication of areas that may be at increased risk of malaria. / AFRIKAANSE OPSOMMING: Alhoewel malaria „n groot publieke gesondheidskwelling bly, veral in sub-Sahara Afrika, bestaan min inligting rakende die fisiologiese toleransies van malaria vektore. Hier het ek gepoog om 'n omvattende reeks van fisiologiese toleransies te voorsien vir Anopheles arabiensis en An. funestus, deur termiese verdraagsaamheidseienskappe, uitdrogingsweerstand en ontwikkelingstempo-temperatuur verhoudings vir beide vektore te ondersoek. Kritiese termiese limiet (CT) en uitdroging data het beduidende uitwerkings getoon van toenemende ouderdom op die vermindering van verdraagsaamheid teenoor temperatuur of droë toestande. Wyfies van beide spesies was meer verdraagsaam vir hoë of lae temperature in CT eksperimente en was meer verdraagsaam teenoor uitdroging as mannetjies in die uitdrogingsproewe. Anopheles funestus was meer verdraagsaam teenoor uitdroging as An. arabiensis, ten spyte van die algemene wanopvatting dat An. arabiensis die meer ariede aangepaste van die twee spesies is. Vergelykings tussen termiese verdraagsaamheidseienskappe van laboratorium-en wilde stamlyn nageslagte van beide spesies het 'n hoë mate van ooreenkoms tussen kritieke termiese limiete in wilde en laboratorium stamlyne aangedui, wat voorstel dat die gebruik van laboratorium bevolkings van beide muskiet stamlyne 'n akkurate skatting kan gee van wilde bevolkingsreaksies tot termiese verandering. Fatale temperatuur beramings vir beide vektore het „n hoër toleransie getoon by hoë temperature in An. arabiensis larwes en papies wanneer dit vergelyk word met An. funestus, en 'n groter verdraagsaamheid van hoë of lae temperature in wyfies, wanneer vergelyk word met mannetjies. Spesies verskille tussen die vektore is verder uitgelig in die ontwikkelingstempo-temperatuur eksperimente. Onder wisselende en konstante temperature ontwikkel An. arabiensis aansienlik vinniger as An. funestus en het hoër oorlewing tot die volwasse stadium getoon. Onder wisselende temperature ontwikkel An. arabiensis vinniger of met geen verskil van konstante temperature nie, terwyl oorlewing onder wisselende temperature ook vergelykbaar was met konstante temperatuur beramings. Die vinniger tempo van hierdie spesie is waarskynlik 'n gevolg van die poel-broeiende aard van An. arabiensis en die behoefte om tot volwassenheid te ontwikkel voor die verdamping van broeiplekke. Anopheles funestus aan die ander kant, het verminderde oorlewing en ontwikkeling onder wisselende temperature gewys wanneer dit vergelyk word met konstante temperature, waarskynlik as gevolg van die meer termies stabiele broeiplekke wat gewoonlik gebruik word deur hierdie spesie. Verspreidingsdata van hierdie spesies, gekombineer met ontwikkelings-parameters in 'n proses-gebaseerde verspreidingsmodel, dui daarop dat beide spesies reeks veranderinge sal wys in reaksie tot klimaatsverandering. Gebiede waar hierdie spesies voorheen slegs teenwoordig was op 'n seisoenale basis, mag dalk meer geskik word vir vektor bevolkingsvestiging en volharding, terwyl areas op die noordelike grense van die huidige verspreidings minder gunstig sal word, wat sal lei tot algehele suidelike verskuiwing in die habitat geskiktheid vir beide spesies. Toenames in temperatuur en veranderinge in reënvalpatrone, soos voorspel word om voor te kom met verandering van die klimaat, sal waarskynlik die verspreiding van malaria vektore beïnvloed. Deur die fisiologiese toleransie data, versamel in hierdie tesis, te kombineer met 'n toekoms, beplande meganistiese verspreidingsmodel, sal dit 'n akkurate aanduiding gee van die potensiële verspreidingsverskuiwings van hierdie vektore en dus 'n aanduiding gee van gebiede wat onder verhoogde risiko van malaria sal wees.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Une distribution nationale de moustiquaires imprégnées d'insecticide à longue durée d'action à destination des populations vulnérables du Niger a été effectuée fin 2005. Déjà montrée lors d'études pilotes à l'échelle du village, l'efficacité de cet outil dans le contrôle du paludisme restait à évaluer à l'occasion de vastes programmes opérationnels qui se multiplient en Afrique. Peu d'études des populations de vecteurs ont été publiées dans ce cadre. Nous avons mis en place un suivi entomologique au niveau de sites sentinelles répartis dans la zone Sahélienne du Niger, ayant couvert trois saisons de transmission, dont une avant intervention considérée comme période contrôle. Les paramètres entomologiques de la transmission ont été déterminés pour An. gambiae s.l., et la distribution spatiale des deux principaux vecteurs, An. gambiae et An. arabiensis, a été précisée. Le suivi temporel a mis en évidence une baisse globale du niveau de transmission de P. falciparum, probablement entrainée par la forte hausse d'utilisation de moustiquaires imprégnées. Cependant la hausse de la résistance des populations aux pyréthrinoïdes semble avoir été rapidement amorcée faisant craindre à moyen terme une perte d'efficacité de cet outil central des stratégies de lutte contre le paludisme. L'étude de la structure génétique des populations d'An. gambiae et d'An. arabiensis à l'aide de marqueurs microsatellites a montré une homogénéité génétique dans l'espace, entre les villages, même séparés par plusieurs centaines de kilomètres, ainsi que dans le temps, entre la saison de transmission 2005 contrôle et la saison 2006 après distribution. Ces résultats ont suggéré qu'au cours de la première année d'intervention, la couverture en moustiquaires imprégnées atteinte n'a pas eu d'effet de masse suffisant pour entrainer une baisse de la diversité génétique ou une modification des fréquences alléliques des populations. La faible différenciation spatiale observée pourrait être expliquée par des échanges de gènes importants à l'intérieur de la zone d'étude, hypothèse appuyée par l'expansion rapide de la mutation kdr dans l'ensemble des sites où An. gambiae est présent. L'évaluation rigoureuse de tels programmes de contrôle permettra d'améliorer les outils de contrôle et par exemple de préserver l'efficacité des pyréthrinoïdes, seule classe d'insecticides actuellement disponible pour l'imprégnation des moustiquaires.

Les femelles moustiques peuvent être vectrices de nombreux agents infectieux (virus, protozoaires, helminthes) pour l'Homme, qui peuvent être la cause de maladies graves comme le paludisme et la dengue. Ces maladies menacent respectivement 50 et 40% de la population mondiale ; le paludisme étant responsable de près d'un million de décès par an. Les méthodes de lutte anti-vectorielle destinées à limiter les populations vectrices et stopper la transmission de maladies, se heurtent au développement incessant de résistances de la part des moustiques et des agents infectieux vis-à-vis des traitements employés. Bien que certaines régions du monde aient réussi à stopper efficacement la transmission de certaines de ces maladies, une grande partie des régions tropicales reste menacée. De plus l'expansion rapide de certaines espèces vectrices, telles qu'Aedes albopictus, accroît les risques sanitaires dans de nouvelles régions du globe. La technique de l'insecte stérile (TIS), qui a permis l'éradication ou la suppression des populations de nombreux insectes nuisibles aux cultures et à l'Homme, représente un moyen de lutte prometteur contre les moustiques. Cette technique s'appuie sur le lâcher en masse de mâles stérilisés par rayonnements ionisants qui, en transférant un sperme stérile aux femelles sauvages, vont permettre une diminution progressive de la population cible. Suite à l'épidémie de chikungunya à l'île de la Réunion en 2005 et face aux menaces permanentes de recrudescence de la dengue et du paludisme, les services de lutte anti-vectorielle réunionnais mettent en place d'importants moyens de lutte contre les populations de moustiques concernées. Toutefois, ces mesures ne permettant pas une diminution durable des densités de vecteurs, une étude de faisabilité est en cours quant à l'utilisation de la TIS pour diminuer et contrôler les populations d'Aedes albopictus, vecteur de la dengue et du chikungunya, et d'Anopheles arabiensis, vecteur du paludisme.Ce travail de thèse s'inscrit dans le cadre du projet TIS Réunion, dans le but d'étudier la biologie et le comportement des souches destinées aux lâchers de mâles stériles. Dans un premier temps, cette étude s'intéresse à la comparaison entre les souches d'élevage d'Anopheles arabiensis et les souches sauvages, ainsi qu'aux modalités de stérilisation des mâles de la souche à sexage génétique. Une seconde partie est consacrée à l'étude de l'effet de l'irradiation sur les mâles d'Aedes albopictus, en étudiant plus particulièrement leur stratégie de reproduction, leur capacité d'insémination en laboratoire, ainsi que leur compétitivité sexuelle et longévité face aux mâles sauvages en conditions semi-contrôlées.