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Background Anopheles arabiensis is a major malaria vector, recently implicated as contributing to ongoing residual malaria transmission in South Africa, which feeds and rests both indoors and outdoors. This species is, therefore, not effectively targeted using core malaria vector control interventions alone. Additionally, increasing resistance to available insecticides necessitates investigations into complementary non-insecticide-based vector control methods for outdoor-resting mosquitoes. The feasibility of the sterile insect technique (SIT) as a complementary vector control intervention is being investigated in South Africa. Successful implementation of an SIT programme largely depends on inundating a target insect population with sterilized laboratory-bred males. Therefore, knowledge of the native population size and dispersal ability of released sterile laboratory-reared males is critical. In this study, we estimated the male An. arabiensis population size and the dispersal of released males in an area targeted for a pilot sterile male release programme. Methods Three separate releases were performed within a 2-year period. Approximately 5000–15,000 laboratory-reared male An. arabiensis (KWAG) were produced and marked for mark–release–recapture experiments. To recapture released mosquitoes, cloth tubes were deployed in widening concentric circles. The average dispersal distance of released males was calculated and the wild male An. arabiensis population size was estimated using two Lincoln index formulae. The natural population was sampled concurrently and Anopheles species diversity examined. Results The Anopheles gambiae complex and An. funestus group species made up the majority of wild collections along with other anophelines. The An. arabiensis population size was estimated to be between 550 and 9500 males per hectare depending on time of year, weather conditions and method used. Average dispersal distance of marked males ranged from 58 to 86 m. Marked males were found in swarms with wild males, indicating that laboratory-reared males are able to locate and participate in mating swarms. Conclusions It was logistically feasible to conduct mark–release–recapture studies at the current scale. The population size estimates obtained may provide a guideline for the initial number of males to use for a pending SIT pilot trial. It is promising for future SIT trials that laboratory-reared marked males participated in natural swarms, appearing at the right place at the right time. Graphical Abstract

The feasibility of the sterile insect technique (SIT) as a malaria vector control strategy against Anopheles arabiensis has been under investigation over the past decade. One of the critical steps required for the application of this technique to mosquito control is the availability of an efficient and effective sex-separation system. Sex-separation systems eliminate female mosquitoes from the production line prior to irradiation and field release of sterile males. This is necessary because female mosquitoes can transmit pathogens such as malaria and, therefore, their release must be prevented. Sex separation also increases the efficiency of an SIT programme. Various sex-separation strategies have been explored including the exploitation of developmental and behavioural differences between male and female mosquitoes, and genetic approaches. Most of these are however species-specific and are not indicated for the major African malaria vectors such as An. arabiensis. As there is currently no reliable sex-separation method for An. arabiensis, various strategies were explored in an attempt to develop a robust system that can be applied on a mass-rearing scale. The progress and challenges faced during the development of a sexing system for future pilot and/or large-scale SIT release programmes against An. arabiensis are reviewed here. Three methods of sex separation were examined. The first is the use of pupal size for gender prediction. The second is the elimination of blood-feeding adult females through the addition of an endectocide to a blood meal source. The third is the establishment of a genetic sexing strain (GSS) carrying an insecticide resistance selectable marker (dieldrin-resistance rdl gene and/or other GABA receptor antagonists that can be used as alternative insecticides to dieldrin) or a temperature-sensitive lethal marker.

Innovations in mosquito mass-rearing techniques are essential in the quest to develop SIT (sterile insect technique) methods to fight mosquito vectors of disease. This study reports modifications to the Food and Agriculture Organisation/International Atomic Energy Agency (FAO/IAEA) mass-rearing cage (MRC) for mosquitoes to support the behaviour of adult Anopheles arabiensis Patton (Diptera: Culicidae) and to maximize egg production. The effects of an improved sugar-feeding device, and the addition of resting sites and a black cloth shroud to create an artificial horizon (visual contrast of light vs. dark at the edge) were assessed for their effect on adult longevity and egg production. Egg production of adults resulting from larvae reared in individual free-standing trays vs. those reared in the same trays in the FAO/IAEA larval rearing rack was also compared. Finally, the effect of blood feeding and frequency of egg collection from the MRC on average egg production per batch was investigated. Overall, the modifications to the MRC enhanced adult longevity, and the improved cage prototype allowed the collection of more eggs overall from a cohort of adults than was possible using the original and previous cage prototypes. These stepwise improvements are important for the development of economical and logistically efficient mass-rearing systems for the malaria vector An. arabiensis.

Malaria vector control by indoor residual spraying (IRS) in South Africa currently relies on the use of DDT to control pyrethroid-resistant populations of the major malaria vector Anopheles funestus. Pyrethroid resistance in An. funestus in South Africa and Mozambique is primarily based on the over-production of monooxygenase P450 genes. Preliminary evidence suggests that the pyrethroid-resistant phenotype is primarily expressed in adults, raising questions as to how resistance was selected for in the field given that there was no malaria control targeting adults in southern Mozambique when this resistant phenotype was first discovered there. The aim of this study therefore was to select for pyrethroid resistance at the larval or adult stages in southern African An. funestus and then assay the pyrethroid susceptibility of subsequent life stages using samples of progeny from the pyrethroid-resistant selections. There was no significant variation between the lethal permethrin concentrations inducing 50% mor...

Background Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. Methods The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. Results Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the highest sterility in the representative wild-type population, with potential to effectively suppress reproduction. Conclusion Laboratory-reared and sterilised GAMA male An. arabiensis at a release ratio of 3:1 (3 sterile males to 1 wild, fertile male) can successfully compete for insemination of wild-type females. These results will be used to inform subsequent small-scale pilot field releases in South Africa.

Background Malaria vector control relies principally on the use of insecticides, especially pyrethroids. Because of the increasing occurrence of insecticide resistance in target vector populations, the development of new insecticides, particularly those with novel modes of action, is particularly important, especially in terms of managing insecticide resistance. The C8910 formulation is a patented mixture of compounds comprising straight-chain octanoic, nonanoic and decanoic saturated fatty acids. This compound has demonstrated toxic and repellent effects against several arthropod species. The aims of this study were to measure the insecticidal effects of C8910 against an insecticide susceptible (FANG) and a pyrethroid resistant (FUMOZ-R) laboratory strain of An. funestus as well as against wild-caught An. funestus material from Zambia (ZamF), and to investigate the repellent effects of two formulations of C8910 against these strains. Methods Toxicity against adult females was assessed using a range of concentrations based on the CDC bottle bioassay method and repellence of three different C8910 formulations was assessed using standard choice-chamber bioassays. Results C8910 proved equally toxic to adult females of the FUMOZ-R and FANG laboratory strains, as well as to adult females of the wild-caught (ZamF) sample. None of the C8910 formulations tested gave any conclusive indication of repellence against any of the strains. Conclusion C8910 is equally effective as an adulticide against pyrethroid resistant and insecticide susceptible An. funestus. However, the formulations tested did not show any consistent repellence against laboratory reared and wild-caught female samples of this species. Nevertheless, C8910 shows potential as an adulticide that can be used for malaria vector control, particularly in those instances where insecticide resistance management is required.

Background Two mitochondrial DNA clades have been described in Anopheles funestus populations from southern Africa. Clade I is common across the continent while clade II is known only from Mozambique and Madagascar. The specific biological status of these clades is at present unknown. We investigated the possible role that each clade might play in the transmission of Plasmodium falciparum and the insecticide resistance status of An. funestus from Zimbabwe and Zambia. Methods Mosquitoes were collected inside houses from Nchelenge District, Zambia and Honde Valley, Zimbabwe in 2013 and 2014. WHO susceptibility tests, synergist assays and resistance intensity tests were conducted on wild females and progeny of wild females. ELISA was used to detect Plasmodium falciparum circumsporozoite protein. Specimens were identified to species and mtDNA clades using standard molecular methods. Results The Zimbabwean samples were all clade I while the Zambian population comprised 80% clade I and 20% clade II in both years of collection. ELISA tests gave an overall infection rate of 2.3% and 2.1% in 2013, and 3.5% and 9.2% in 2014 for Zimbabwe and Zambia respectively. No significant difference was observed between the clades. All populations were resistant to pyrethroids and carbamates but susceptible to organochlorines and organophosphates. Synergist assays indicated that pyrethroid resistance is mediated by cytochrome P450 mono-oxygenases. Resistance intensity tests showed high survival rates after 8-hrs continuous exposure to pyrethroids but exposure to bendiocarb gave the same results as the susceptible control. Conclusions This is the first record of An. funestus mtDNA clade II occurring in Zambia. No evidence was found to suggest that the clades are markers of biologically separate populations. The ability of An. funestus to withstand prolonged exposure to pyrethroids has serious implications for the use of these insecticides, either through LLINs or IRS, in southern Africa in general and resistance management strategies should be urgently implemented.