South Africa aims to eliminate malaria within its borders by 2018. Despite well-coordinated provincial vector control programmes that are based on indoor residual insecticide spraying, low-level residual malaria transmission continues in the low-altitude border regions of the north-eastern sector of the country. In order to identify the underlying causes of residual transmission, an enhanced vector surveillance system has been implemented at selected sites in the Mpumalanga and KwaZulu-Natal (KZN) provinces. The collection periods for the data presented are March 2015 to April 2016 for Mpumalanga and January 2014 to December 2015 for KZN. The mosquito collection methods used included indoor and outdoor traps based on the use of traditional ceramic pots, modified plastic buckets and exit window traps (KZN only). All Anopheles funestus species group mosquitoes collected were identified to species and all females were screened for the presence of Plasmodium falciparum sporozoites. Two An. vaneedeni females, one from each surveillance site, tested positive for P. falciparum sporozoites. These are the first records of natural populations of An. vaneedeni being infective with P. falciparum. As both specimens were collected from outdoor-placed ceramic pots, these data show that An. vaneedeni likely contributes to residual malaria transmission in South Africa.

Background: Programmatic monitoring of insecticide resistance in disease vectors is mostly done on a large scale, often focusing on differences between districts, regions or countries. However, local heterogeneities in residual malaria transmission imply the need for finer-scale data. This study reports small-scale variations of insecticide susceptibility in Anopheles arabiensis between three neighbouring villages across two seasons in Tanzania, where insecticidal bed nets are extensively used, but malaria transmission persists. Methods: WHO insecticide susceptibility assays were conducted on female and male An. arabiensis from three proximal villages, Minepa, Lupiro, and Mavimba, during dry (June-December 2015) and wet (January-May 2016) seasons. Adults emerging from wild-collected larvae were exposed to 0.05% lambda-cyhalothrin, 0.05% deltamethrin, 0.75% permethrin, 4% DDT, 4% dieldrin, 0.1% bendiocarb, 0.1% propoxur, 0.25% pirimiphos-methyl and 5% malathion. A hydrolysis probe assay was used to screen for L1014F (kdr-w) and L1014S (kdr-e) mutations in specimens resistant to DDT or pyrethroids. Synergist assays using piperonly butoxide (PBO) and triphenol phosphate (TPP) were done to assess pyrethroid and bendiocarb resistance phenotypes. Results: There were clear seasonal and spatial fluctuations in phenotypic resistance status in An. arabiensis to pyrethroids, DDT and bendiocarb. Pre-exposure to PBO and TPP, resulted in lower knockdown rates and higher mortalities against pyrethroids and bendiocarb, compared to tests without the synergists. Neither L1014F nor L1014S mutations were detected. Conclusions: This study confirmed the presence of pyrethroid resistance in An. arabiensis and showed small-scale differences in resistance levels between the villages, and between seasons. Substantial, though incomplete, reversal of pyrethroid and bendiocarb resistance following pre-exposure to PBO and TPP, and absence of kdr alleles suggest involvement of P450 monooxygenases and esterases in the resistant phenotypes. We recommend, for effective resistance management, further bioassays to quantify the strength of resistance, and both biochemical and molecular analysis to elucidate specific enzymes responsible in resistance.

Background: Insecticide resistance can present a major obstacle to malaria control programmes. Following the recent detection of DDT resistance in Anopheles arabiensis in Gokwe, Zimbabwe, the underlying resistance mechanisms in this population were studied. Methods: Standard WHO bioassays, using 0.75% permethrin, 4% DDT, 5% malathion, 0.1% bendiocarb and 4% dieldrin were performed on wild-collected adult anopheline mosquitoes and F₁ progeny of An. arabiensis reared from wild-caught females. Molecular techniques were used for species identification as well as to identify knockdown resistance (kdr) and ace-1 mutations in individual mosquitoes. Biochemical assays were used to determine the relative levels of detoxifying enzyme systems including non-specific esterases, monooxygenases and glutathione-S-transferases as well as to detect the presence of an altered acetylcholine esterase (AChE). Results: Anopheles arabiensis was the predominant member of the Anopheles gambiae complex. Of the 436 An. arabiensis females, 0.5% were positive for Plasmodium falciparum infection. WHO diagnostic tests on wild populations showed resistance to the pyrethroid insecticide permethrin at a mean mortality of 47% during February 2006 and a mean mortality of 68.2% in January 2008. DDT resistance (68.4% mean mortality) was present in February 2006; however, two years later the mean mortality was 96%. Insecticide susceptibility tests on F₁ An. arabiensis families reared from material from two separate collections showed an average mean mortality of 87% (n = 758) after exposure to 4% DDT and 65% (n = 587) after exposure to 0.75% permethrin. Eight families were resistant to both DDT and permethrin. Biochemical analysis of F₁ families reared from collections done in 2006 revealed high activity levels of monooxygenase (48.5% of families tested, n = 33, p < 0.05), glutathione S-transferase (25.8% of families tested, n = 31, p < 0.05) and general esterase activity compared to a reference susceptible An. arabiensis colony. Knockdown resistance (kdr) and ace-I ^R mutations were not detected. Conclusion: This study confirmed the presence of permethrin resistance in An. arabiensis populations from Gwave and emphasizes the importance of periodic and ongoing insecticide susceptibility testing of malaria vector populations whose responses to insecticide exposure may undergo rapid change over time.

Malaria control in Africa relies heavily on indoor vector management, primarily indoor residual spraying and insecticide treated bed nets. Little is known about outdoor biting behaviour or even the dynamics of indoor biting and infection risk of sleeping household occupants. In this paper we explore the preferred biting sites on the human body and some of the ramifications regarding infection risk and exposure management. We undertook whole-night human landing catches of Anopheles arabiensis in South Africa and Anopheles gambiae s.s. and Anopheles funestus in Uganda, for seated persons wearing short sleeve shirts, short pants, and bare legs, ankles and feet. Catches were kept separate for different body regions and capture sessions. All An. gambiae s.l. and An. funestus group individuals were identified to species level by PCR. Three of the main vectors of malaria in Africa (An. arabiensis, An. gambiae s.s. and An. funestus) all have a preference for feeding close to ground level, which is manifested as a strong propensity (77.3% – 100%) for biting on lower leg, ankles and feet of people seated either indoors or outdoors, but somewhat randomly along the lower edge of the body in contact with the surface when lying down. If the lower extremities of the legs (below mid-calf level) of seated people are protected and therefore exclude access to this body region, vector mosquitoes do not move higher up the body to feed at alternate body sites, instead resulting in a high (58.5% - 68.8%) reduction in biting intensity by these three species. Protecting the lower limbs of people outdoors at night can achieve a major reduction in biting intensity by malaria vector mosquitoes. Persons sleeping at floor level bear a disproportionate risk of being bitten at night because this is the preferred height for feeding by the primary vector species. Therefore it is critical to protect children sleeping at floor level (bednets; repellent-impregnated blankets or sheets, etc.). Additionally, the opportunity exists for the development of inexpensive repellent-impregnated anklets and/or sandals to discourage vectors feeding on the lower legs under outdoor conditions at night.

Background: The successful suppression of a target insect population using the sterile insect technique (SIT) partly depends on the premise that the laboratory insects used for mass rearing are genetically compatible with the target population, that the mating competitiveness of laboratory reared males is at least comparable to that of their wild counterparts, and that mass rearing and sterilization processes do not in themselves compromise male fitness to a degree that precludes them from successfully competing for mates in the wild. This study investigated the fitness and sexual cross-compatibility between samples of field collected and laboratory reared An. arabiensis under laboratory conditions. Results: The physiological and reproductive fitness of the MALPAN laboratory strain is not substantially modified with respect to the field population at Malahlapanga. Further, a high degree of mating compatibility between MALPAN and the Malahlapanga population was established based on cross-mating experiments. Lastly, the morphological characteristics of hybrid ovarian polytene chromosomes further support the contention that the MALPAN laboratory colony and the An. arabiensis population at Malahlapanga are genetically homogenous and therefore compatible. Conclusions: It is concluded that the presence of a perennial and isolated population of An. arabiensis at Malahlapanga presents a unique opportunity for assessing the feasibility of SIT as a malaria vector control option. The MALPAN laboratory colony has retained sufficient enough measures of reproductive and physiological fitness to present as a suitable candidate for male sterilization, mass rearing and subsequent mass release of sterile males at Malahlapanga in order to further assess the feasibility of SIT in a field setting.

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. 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)]. 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. 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.