Optimisation of laboratory-rearing parameters for Anopheles funestus larvae and adults.

Anopheles funestus is one of the major malaria vectors in Africa. As with the other main vectors, insecticide resistance in this species threatens existing vector control strategies. Unfortunately, scientific investigations, which could improve understanding of this vector species or lead to the development of new control strategies, are currently limited by difficulties in laboratory rearing of the species. In an attempt to optimise laboratory-rearing conditions for An. funestus, the effect of an artificial blood-feeding system for adults, different larval diet doses, and a range of other rearing conditions on the life history traits of an existing colony were investigated. Firstly, fecundity and fertility in An. funestus adult females fed on either live guinea pigs or bovine blood supplied through an artificial membrane feeding system were assessed. Secondly, a life-table approach was used to assess the impact of larval food dose (mg/larvae), larval density (larvae/cm2), and the depth of water used for larval rearing on life history traits. Fecundity was significantly higher when females were blood-fed on live anaesthetised guinea pigs than when fed on defibrinated bovine blood. However, the fertility of these eggs did not differ significantly between the two feeding methods or blood meal sources. Mosquitoes fed on defibrinated bovine blood using the artificial membrane feeding system showed an increase in egg production when the blood-feeding frequency was increased, but this difference was not statistically significant. The quantity of larval food influenced both time-to-pupation and pupal production. Increasing the larval densities resulted in reduced both time-to-pupation and pupal productivity. An optimal larval density of 0.48 larvae/cm2 was vital in preventing overcrowding. Increased water depth in the larval trays, was associated with significantly lower pupal production and reduced pupal weight. In conclusion, these results show that An. funestus can be reared using defibrinated bovine blood delivered via an artificial membrane feeding system. The quantity of larval food, optimal larval density, and depth of water used for larval rearing are critical factors influencing colony productivity. These findings can be used to improve current guidelines for rearing An. funestus under insectary conditions.

Life-history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background.

BACKGROUND: South Africa has set a mandate to eliminate local malaria transmission by 2023. In pursuit of this objective a Sterile Insect Technique programme targeting the main vector Anopheles arabiensis is currently under development. Significant progress has been made towards operationalizing the technology. However, one of the main limitations being faced is the absence of an efficient genetic sexing system. This study is an assessment of an An. arabiensis (AY-2) strain carrying the full Y chromosome from Anopheles gambiae, including a transgenic red fluorescent marker, being introgressed into a South African genetic background as a potential tool for a reliable sexing system. METHODS: Adult, virgin males from the An. arabiensis AY-2 strain were outcrossed to virgin females from the South African, Kwazulu-Natal An. arabiensis (KWAG strain) over three generations. Anopheles arabiensis AY-2 fluorescent males were sorted as first instar larvae (L1) using the Complex Object Parametric Analyzer and Sorter (COPAS) and later screened as pupae to verify the sex. Life history traits of the novel hybrid KWAG-AY2 strain were compared to the original fluorescent AY-2 strain, the South African wild-type KWAG strain and a standard laboratory An. arabiensis (Dongola reference strain). RESULTS: The genetic stability of the sex-linked fluorescent marker and the integrity and high level of sexing efficiency of the system were confirmed. No recombination events in respect to the fluorescent marker were detected over three rounds of introgression crosses. KWAG-AY2 had higher hatch rates and survival of L1 to pupae and L1 to adult than the founding strains. AY-2 showed faster development time of immature stages and larger adult body size, but lower larval survival rates. Adult KWAG males had significantly higher survival rates. There was no significant difference between the strains in fecundity and proportion of males. KWAG-AY2 males performed better than reference strains in flight ability tests. CONCLUSION: The life history traits of KWAG-AY2, its rearing efficiency under laboratory conditions, the preservation of the sex-linked fluorescence and perfect sexing efficiency after three rounds of introgression crosses, indicate that it has potential for mass rearing. The potential risks and benefits associated to the use of this strain within the Sterile Insect Technique programme in South Africa are discussed.

Effect of larval density and additional anchoring surface on the life-history traits of a laboratory colonized Anopheles funestus strain.

Optimal rearing conditions, inclusive of larval rearing density, are critical for sustained mosquito productivity. There is limited information on favourable conditions for the larval rearing of Anopheles funestus, the dominant malaria vector in east and southern Africa. This work investigated the effects of larval rearing densities and additional anchoring surface on An. funestus development using a life table approach. Larval cohorts were reared at four different larval densities using the same rearing surface area, larval food concentrations and temperature conditions. Rearing larvae at high densities extended the larval developmental time and reduced adult productivity. Adding an extra larval anchoring surface when rearing larvae at high density resulted in extended larval developmental time, increased larval survivorship and produced bigger adults. These findings improve our understanding of the relationship between larval density and developmental traits in An. funestus and provides baseline information for An. funestus rearing under laboratory conditions.

"Maskandi experience": exploring the use of a cultural song for community engagement in preparation for a pilot Sterile Insect Technique release programme for malaria vector control in KwaZulu-Natal Province, South Africa 2019.

BACKGROUND: An assessment of the Sterile Insect Technique (SIT) as a complementary malaria vector control tool, is at an advanced stage in South Africa. The technique involves the release of laboratory-reared sterilized male mosquitoes of the major malaria vector Anopheles arabiensis, raising social, ethical and regulatory concerns. Therefore, its implementation largely depends on community participation and acceptance. Against this background, it is critical that robust and effective community strategies are developed. This study describes the development of a cultural song to engage the community and increase awareness on SIT and malaria control in KwaZulu-Natal, South Africa. METHODS: An exploratory concurrent mixed-methods study was conducted to get opinions about the effectiveness of a cultural song developed to engage communities and increase acceptability of the SIT technology. Two self-administered surveys (expert and community) were conducted. Additionally, more in depth opinions of the song and its effectiveness in conveying the intended information were investigated through three community dialogue sessions with community members in the study area. RESULTS: A total of 40 experts and 54 community members participated in the survey. Four themes were identified in relation to the appropriateness and effectiveness of the song, with a fifth theme focused on recommendations for adaptations. Overall, the song was well received with the audience finding it entertaining and informative. Responses to unstructured questions posed after the song showed an increase in the knowledge on malaria transmission and SIT technology. In particular, the explanation that male mosquitoes do not bite allayed anxiety and fears about the SIT technology. CONCLUSION: The song was deemed both culturally appropriate and informative in engaging community members about the SIT technology. It proved useful in promoting health messages and conveying SIT technology as a complementary malaria vector control tool. With minor adaptations, the song has potential as an area-wide community engagement tool in areas targeted for sterile male releases.

Estimates of the population size and dispersal range of Anopheles arabiensis in Northern KwaZulu-Natal, South Africa: implications for a planned pilot programme to release sterile male mosquitoes.

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.

Characterization of life-history parameters of an Anopheles funestus (Diptera: Culicidae) laboratory strain.

The colonization of the African malaria vector Anopheles funestus has been hampered by inadequate knowledge of its mating and development under laboratory conditions. Life-tables are routinely used to provide baseline biological characteristics needed for colonization. This study characterized age-specific life-table attributes of an existing An. funestus laboratory strain to gain insight into factors that are critical for its colonization. To achieve this, the An. funestus laboratory strain was reared from eggs to adulthood under standard insectary conditions, monitoring and characterizing each developmental stage. The mean insemination rate of females was 74.8% with an average egg load of 67.1 eggs/female and a mean fertility of 86.7%. The mean developmental time from 1st instar larvae (L1) to pupation was 16.4 days. The mean proportion of L1 that survived to pupation was 72.9%. On average, 78.8% of the pupae successfully eclosed as adults. The median longevity for adult males and females was 44 and 28 days, respectively. This work constitutes the first report on life-table characterization of an An. funestus strain. The larval developmental time was within the range reported for wild An. funestus while adult longevity was higher compared to survivorship observed in wild populations. These data demonstrate that the colonized An. funestus strain has potential to be re-colonized under standard insectary conditions. The study provides base-line information for further studies on identifying critical parameters for the maintenance of An. funestus under artificial conditions.

Patterns of pesticide usage in agriculture in rural Tanzania call for integrating agricultural and public health practices in managing insecticide-resistance in malaria vectors.

BACKGROUND: Unrestricted use of pesticides in agriculture is likely to increase insecticide resistance in mosquito vectors. Unfortunately, strategies for managing insecticide resistance in agriculture and public health sectors lack integration. This study explored the types and usage of agricultural pesticides, and awareness and management practices among retailers and farmers in Ulanga and Kilombero districts in south-eastern Tanzania, where Anopheles mosquitoes are resistant to pyrethroids. METHODS: An exploratory sequential mixed-methods approach was employed. First, a survey to characterize pesticide stocks was conducted in agricultural and veterinary (agrovet) retail stores. Interviews to assess general knowledge and practices regarding agricultural pesticides were performed with 17 retailers and 30 farmers, followed by a survey involving 427 farmers. Concurrently, field observations were done to validate the results. RESULTS: Lambda-cyhalothrin, cypermethrin (both pyrethroids) and imidacloprids (neonicotinoids) were the most common agricultural insecticides sold to farmers. The herbicide glyphosate (amino-phosphonates) (59.0%), and the fungicides dithiocarbamate and acylalanine (54.5%), and organochlorine (27.3%) were also readily available in the agrovet shops and widely used by farmers. Although both retailers and farmers had at least primary-level education and recognized pesticides by their trade names, they lacked knowledge on pest control or proper usage of these pesticides. Most of the farmers (54.4%, n = 316) relied on instructions from pesticides dealers. Overall, 93.7% (400) farmers practised pesticides mixing in their farms, often in close proximity to water sources. One-third of the farmers disposed of their pesticide leftovers (30.0%, n = 128) and most farmers discarded empty pesticide containers into rivers or nearby bushes (55.7%, n = 238). CONCLUSION: Similarities of active ingredients used in agriculture and malaria vector control, poor pesticide management practices and low-levels of awareness among farmers and pesticides retailers might enhance the selection of insecticide resistance in malaria vectors. This study emphasizes the need for improving awareness among retailers and farmers on proper usage and management of pesticides. The study also highlights the need for an integrated approach, including coordinated education on pesticide use, to improve the overall management of insecticide resistance in both agricultural and public health sectors.

High levels of imported asymptomatic malaria but limited local transmission in KwaZulu-Natal, a South African malaria-endemic province nearing malaria elimination.

BACKGROUND: KwaZulu-Natal, one of South Africa's three malaria endemic provinces, is nearing malaria elimination, reporting fewer than 100 locally-acquired cases annually since 2010. Despite sustained implementation of essential interventions, including annual indoor residual spraying, prompt case detection using malaria rapid diagnostics tests and treatment with effective artemisinin-based combination therapy, low-level focal transmission persists in the province. This malaria prevalence and entomological survey was therefore undertaken to identify the drivers of this residual transmission. METHODS: Malaria prevalence as well as malaria knowledge, attitudes and practices among community members and mobile migrant populations within uMkhanyakude district, KwaZulu-Natal were assessed during a community-based malaria prevalence survey. All consenting participants were tested for malaria by both conventional and highly-sensitive falciparum-specific rapid diagnostic tests. Finger-prick filter-paper blood spots were also collected from all participants for downstream parasite genotyping analysis. Entomological investigations were conducted around the surveyed households, with potential breeding sites geolocated and larvae collected for species identification and insecticide susceptibility testing. A random selection of households were assessed for indoor residual spray quality by cone bioassay. RESULTS: A low malaria prevalence was confirmed in the study area, with only 2% (67/2979) of the participants found to be malaria positive by both conventional and highly-sensitive falciparum-specific rapid diagnostic tests. Malaria prevalence however differed markedly between the border market and community (p < 0001), with the majority of the detected malaria carriers (65/67) identified as asymptomatic Mozambican nationals transiting through the informal border market from Mozambique to economic hubs within South Africa. Genomic analysis of the malaria isolates revealed a high degree of heterozygosity and limited genetic relatedness between the isolates supporting the hypothesis of limited local malaria transmission within the province. New potential vector breeding sites, potential vector populations with reduced insecticide susceptibility and areas with sub-optimal vector intervention coverage were identified during the entomological investigations. CONCLUSION: If KwaZulu-Natal is to successfully halt local malaria transmission and prevent the re-introduction of malaria, greater efforts need to be placed on detecting and treating malaria carriers at both formal and informal border crossings with transmission blocking anti-malarials, while ensuring optimal coverage of vector control interventions is achieved.

Fine-scale spatial and temporal variations in insecticide resistance in Culex pipiens complex mosquitoes in rural south-eastern Tanzania.

BACKGROUND: Culex mosquitoes cause considerable biting nuisance and sporadic transmission of arboviral and filarial diseases. METHODS: Using standard World Health Organization procedures, insecticide resistance profiles and underlying mechanisms were investigated during dry and wet seasons of 2015 and 2016 in Culex pipiens complex from three neighbouring administrative wards in Ulanga District, Tanzania. Synergist tests with piperonyl butoxide, diethyl maleate, and triphenyl phosphate, were employed to investigate mechanisms of the observed resistance phenotypes. Proportional biting densities of Culex species, relative to other taxa, were determined from indoor surveillance data collected in 2012, 2013, and 2015. RESULTS: Insecticide resistance varied significantly between wards and seasons. For example, female mosquitoes in one ward were susceptible to bendiocarb and fenitrothion in the wet season, but resistant during the dry season, while in neighbouring ward, the mosquitoes were fully susceptible to these pesticides in both seasons. Similar variations occurred against bendiocarb, DDT, deltamethrin, and lambda-cyhalothrin. Surprisingly, with the exception of one ward in the wet season, the Culex populations were susceptible to permethrin, commonly used on bednets in the area. No insecticide resistance was observed against the organophosphates, pirimiphos-methyl and malathion, except for one incident of reduced susceptibility in the dry season. Synergist assays revealed possible involvement of monooxygenases, esterases, and glutathione S-transferase in pyrethroid and DDT resistance. Morphology-based identification and molecular assays of adult Culex revealed that 94% were Cx. pipiens complex, of which 81% were Cx. quinquefasciatus, 2% Cx. pipiens, and 3% hybrids. About 14% of the specimens were non-amplified during molecular identifications. Female adults collected indoors were 100% Cx. pipiens complex, and constituted 79% of the overall biting risk. CONCLUSIONS: The Cx. pipiens complex constituted the greatest biting nuisance inside people's houses, and showed resistance to most public health insecticides possible. Resistance varied at a fine geographical scale, between adjacent wards, and seasons, which warrants some modifications to current insecticide resistance monitoring strategies. Resistance phenotypes are partly mediated by metabolic mechanisms, but require further evaluation through biochemical and molecular techniques. The high densities and resistance in Culex could negatively influence the acceptability of other interventions such as those used against malaria mosquitoes.

A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis.

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.

Population Dynamics and Plasmodium falciparum (Haemosporida: Plasmodiidae) Infectivity Rates for the Malaria Vector Anopheles arabiensis (Diptera: Culicidae) at Mamfene, KwaZulu-Natal, South Africa.

Anopheles arabiensis (Patton; Diptera: Culicidae) is a major malaria vector in the southern African region. In South Africa, effective control of this species using indoor-based interventions is reduced owing to its tendency to rest outdoors. As South Africa moves towards malaria elimination there is a need for complementary vector control strategies. One of the methods under consideration is the use of the sterile insect technique (SIT). Key to the successful implementation of an SIT programme is prior knowledge of the size and spatial distribution of the target population. Understanding mosquito population dynamics for both males and females is critical for efficient programme implementation. It is thus necessary to use outdoor-based population monitoring tools capable of sampling both sexes of the target population. In this project mosquito surveillance and evaluation of tools capable of collecting both genders were carried out at Mamfene in northern KwaZulu-Natal Province, South Africa, during the period January 2014 to December 2015. Outdoor- and indoor-resting Anopheles mosquitoes were sampled in three sections of Mamfene over the 2-yr sampling period using modified plastic buckets, clay pots and window exit traps. Morphological and molecular techniques were used for species identifications of all samples. Wild-caught adult females were tested for Plasmodium falciparum (Welch; Haemosporida: Plasmodiidae) infectivity. Out of 1,705 mosquitoes collected, 1,259 (73.8%) and 255 (15%) were identified as members of either the Anopheles gambiae complex or Anopheles funestus group respectively. An. arabiensis was the most abundant species contributing 78.8% of identified specimens. Mosquito density was highest in summer and lowest during winter. Clay pots yielded 16.3 mosquitoes per trap compared to 10.5 for modified plastic buckets over the 2-yr sampling period. P. falciparum infection rates for An. arabiensis were 0.7% and 0.5% for 2014 and 2015, respectively. Logistic regression analysis showed an association between An. arabiensis catches with Section and season of collection but not with sex and collection methods. These data confirmed the presence of a perennial An. arabiensis population at Mamfene and constitute the first records of P. falciparum infective An. arabiensis from South Africa, confirming this species as a major vector in the malaria endemic provinces of the country.

Effect of ionising (gamma) radiation on female Anopheles arabiensis.

Background: In a mosquito sterile insect technique programme the ideal scenario is to release male mosquitoes only. However, because there are currently no sex separation strategies which guarantee total female elimination, this study investigated the effect of irradiation on physiological and reproductive fitness of females of an Anopheles arabiensis genetic sexing strain. Methods: Female pupae were irradiated at 70 Gy and the effects of irradiation on adult emergence, longevity, blood-feeding capability, mating ability, fecundity and fertility were assessed. Results and conclusion: Irradiation reduced adult emergence and fecundity but did not affect adult survivorship, mating and blood feeding ability, which suggests that irradiated female mosquitoes can transmit disease pathogens.

A new malaria vector mosquito in South Africa.

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.

Fine-scale spatial and temporal heterogeneities in insecticide resistance profiles of the malaria vector, Anopheles arabiensis in rural south-eastern Tanzania.

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.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

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.

Biting behaviour of African malaria vectors: 1. where do the main vector species bite on the human body?

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.

Field study site selection, species abundance and monthly distribution of anopheline mosquitoes in the northern Kruger National Park, South Africa.

BACKGROUND: Knowledge of the ecology and behaviour of a target species is a prerequisite for the successful development of any vector control strategy. Before the implementation of any strategy it is essential to have comprehensive information on the bionomics of species in the targeted area. The aims of this study were to conduct regular entomological surveillance and to determine the relative abundance of anopheline species in the northern Kruger National Park. In addition to this, the impact of weather conditions on an Anopheles arabiensis population were evaluated and a range of mosquito collection methods were assessed. METHODS: A survey of Anopheles species was made between July 2010 and December 2012. Mosquitoes were collected from five sites in the northern Kruger National Park, using carbon dioxide-baited traps, human landing and larval collections. Specimens were identified morphologically and polymerase chain reaction assays were subsequently used where appropriate. RESULTS: A total of 3,311 specimens belonging to nine different taxa was collected. Species collected were: Anopheles arabiensis (n = 1,352), Anopheles quadriannulatus (n = 870), Anopheles coustani (n = 395), Anopheles merus (n = 349), Anopheles pretoriensis (n = 35), Anopheles maculipalpis (n = 28), Anopheles rivulorum (n = 19), Anopheles squamosus (n = 3) and Anopheles rufipes (n = 2). Members of the Anopheles gambiae species complex were the most abundant and widely distributed, occurring across all collection sites. The highest number of mosquitoes was collected using CO2 baited net traps (58.2%) followed by human landing catches (24.8%). Larval collections (17%) provided an additional method to increase sample size. Mosquito sampling productivity was influenced by prevailing weather conditions and overall population densities fluctuated with seasons. CONCLUSION: Several anopheline species occur in the northern Kruger National Park and their densities fluctuate between seasons. Species abundance and relative proportions within the An. gambiae complex varied between collection methods. There is a perennial presence of an isolated population of An. arabiensis at the Malahlapanga site which declined in density during the dry winter months, making this site suitable for a small pilot study site for Sterile Insect Technique as a malaria vector control strategy.

Evaluating the potential of the sterile insect technique for malaria control: relative fitness and mating compatibility between laboratory colonized and a wild population of Anopheles arabiensis from the Kruger National Park, South Africa.

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.

Pyrethroid resistance in the major malaria vector Anopheles arabiensis from Gwave, a malaria-endemic area in Zimbabwe.

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-IR 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.