Characterisation of resistance mechanisms in the major malaria vector Anopheles arabiensis from southern Africa

The continual increase in insecticide resistance in malaria vector mosquitoes is threatening the efficacy and sustainability of most malaria control programmes. The major problem facing malaria vector control programmes is lack of scientific information to assist in resistance management strategies. The objective of this thesis is to elucidate insecticide resistance mechanisms in Anopheles arabiensis from southern Africa. This information is necessary to predict cross resistance spectrum, facilitate choosing alternative insecticides and assist in mapping areas with resistant populations. Field studies to update data available on insecticide susceptibility and characterise the resistance mechanisms were carried out in Gokwe, a malaria endemic area in Zimbabwe. In addition this study reports changes in insecticide resistance levels, detoxification enzymes and P450 gene profile following artificial permethrin and bendiocarb selection of laboratory colonies originating from Mamfene, South Africa. A total of 943 anophelines belonging to four different taxa were collected over a two year period with the majority (98.8%) being members of the An. gambiae complex. Species in the An. gambiae complex were identified by polymerase chain reaction (PCR) and An. arabiensis (72.0%) predominated over all the other sibling species. Among the An. arabiensis females 0.5% and 4.8% was positive for Plasmodium falciparum in 2006 and 2008 respectively. WHO diagnostic tests on wild Anopheles arabiensis populations showed resistance to permethrin at a mean mortality of 47% during 2006 and a mean mortality of 69.4% in 2008. DDT resistance (68.4% mean mortality) was present in 2006; however in 2008 a mean mortality of 94.8% was recorded. Insecticide susceptibility tests on F1 An. arabiensis families showed that 25.4% (n = 59) and 14.2% (n = 14) of the families were resistant to DDT in 2006 and 2008 respectively. For permethrin exposures 56.8% (n = 37) and 78.8% (n = 14) families were resistant in 2006 and 2008 respectively. Eight families were resistance to both DDT and permethrin during the two collection periods. Biochemical assays of F1 An. arabiensis families reared from 2006 collections revealed comparatively high levels of monooxygenase (48%.5% of families tested, n = 33, p<0.05), glutathione S-transferase (26.7% of families tested, n = 30, p<0.05) and general esterases activity compared to the reference colony. No knockdown resistance (kdr) and ace-IR mutations were found. After 12 generations of bendiocarb and permethrin selections of An. arabiensis mosquitoes from Kwazulu/Natal there was approximately a 2.8-fold and 3.8-fold respective increase in the LT50 compared to the parental colony. Selections resulted in increased levels of non-specific esterase and monooxygenase activity for the permethrin selected cohorts, and elevated glutathione S-transferases and general esterases for the bendiocarb colonies. Involvement of monooxygenase and glutathione S-transferase in pyrethroid and bendiocarb resistance was confirmed by synergist studies using piperonyl butoxide and diethyl maleate respectively. P450 gene profiling of the permethrin selected line showed that 4 genes CYP6Z1 (4.67-fold), CYP6Z2 (1.72-fold), CYP6M2 (2.24-fold) and CYP4G16 (1.39-fold) were over expressed in the resistant cohorts. Continued use of pyrethroid insecticides by the Zimbabwean National Malaria Control programme (NMCP) is likely to further select resistant vectors. Use of insecticides such as organophosphates and carbamates, mosaic insecticides or rotation of insecticides is recommended. These results contribute to our limited knowledge of metabolic resistance mechanisms and provide useful information for future studies to come up with new insecticides and diagnostic tools for detection and management of resistance

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

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.The National Research Foundation and by the International Atomic Energy Agency (Contract no SAF16780 (under the G34002) and CRPG34002).http://www.malariajournal.com/am201

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.Bill & Melinda Gates Foundation through the Grand Challenges Explorations initiative.http://www.parasitesandvectors.comhb201

Malaria risk and receptivity: Continuing development of insecticide resistance in the major malaria vector Anopheles arabiensis in northern KwaZulu-Natal, South Africa

Malaria incidence in South Africa is highest in the three endemic provinces: KwaZulu-Natal, Mpumalanga and Limpopo. The contribution to malaria transmission by several mosquito species, variation in their resting behaviours and low levels of insecticide resistance makes it necessary to periodically monitor Anopheles species assemblages and resistance phenotypes in vector populations. The aim of this study was therefore to assess Anopheles species assemblage in northern KwaZulu-Natal and to collect insecticide susceptibility data for An. arabiensis, the primary vector of malaria in that province. Anopheles specimens were collected from Mamfene, Jozini, northern KwaZulu-Natal from November 2019 to April 2021. Progeny of wild-collected An. arabiensis females were used for standard insecticide susceptibility tests and synergist bioassays. Anopheles arabiensis contributed 85.6% (n=11 062) of the total catches. Samples for subsequent insecticide susceptibility bioassays were selected from 212 An. arabiensis families. These showed low-level resistance to DDT, permethrin, deltamethrin, and bendiocarb, as well as full susceptibility to pirimiphos-methyl. Synergist bioassays using piperonyl butoxide and triphenyl phosphate suggest oxygenase-based pyrethroid and esterase-mediated sequestration of bendiocarb. These low levels of resistance are unlikely to be operationally significant at present. It is concluded that northern KwaZulu-Natal Province remains receptive to malaria transmission despite ongoing control and elimination interventions. This is due to the perennial presence of the major vector An. arabiensis and other secondary vector species. The continued detection of low-frequency insecticide resistance phenotypes in An. arabiensis is cause for concern and requires periodic monitoring for changes in resistance frequency and intensity.Significance:• Insecticide resistance in the major malaria vector Anopheles arabiensis in northern KwaZulu-Natal Province is cause for concern in terms of resistance management and ongoing vector control leading toward malaria elimination.• Despite ongoing control interventions, northern KwaZulu-Natal remains receptive to malaria owing to the perennial presence of several Anopheles vector species

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

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.The Nuclear Technologies in Medicine and the Bioscience Initiatives (NTeMBI), a national platform developed and managed by the South African Nuclear Energy Corporation and supported by the Department of Science and Technology. Funding was also provided in part from the National Research Foundation, the International Atomic Energy Agency (Contracts 17904, SAF5013 and SAF16780/ under the G34002) and a Global Diseases Detection/CDC grant (U19GH000622-01 MAL01).http://www.parasitesandvectors.comam2016Paraclinical Science

Malaria Vector Status and Insecticide Susceptibility Studies in Zimbabwe

The major problem that has evolved from malaria control progammes that utilize insecticides for vector control is the development of insecticide resistance. In Zimbabwe, despite decades of pesticide usage there is limited information on susceptibility status of major malaria vectors and possible mechanisms responsible for resistance. Susceptibility status of vectors provides useful information for making rational decisions on control strategies. The malaria vector status and their susceptibility to insecticides were studied in Gokwe, Gwave village in the Midlands province of Zimbabwe. Standard WHO bioassays, using 0.75% permethrin, 4% DDT, 5% malathion, 0.1% bendiocarb and 4% dieldrin were done on wild-collected adult anopheline mosquitoes. Susceptibility tests were done on the F1 generation of Anopheles arabiensis reared from wild-caught females using 0.75% permethrin and 4% DDT. Molecular and biochemical assays were carried out to identify kdr mutations in individual mosquitoes and to determine expression levels of non-specific esterases, monooxygenases, glutathione-S-transferases and altered acetylcholinesterase (AChE). A total of 648 anophelines were collected, with the majority (98%) being members of the An. gambiae complex. Species in the An. gambiae complex were identified by polymerase chain reaction (PCR) and An. arabiensis (72.8%) predominated all the other sibling species. Among the An. arabiensis females, 0.5% was positive for Plasmodium falciparum. WHO diagnostic tests showed that 53% of the An. arabiensis were resistant to permethrin and 32% to DDT. Insecticide susceptibility tests on F1 An. arabiensis families showed an average mortality of 85.9% (n=567) after exposure to 4% DDT and 69.8% (n=372) after exposure to 0.75% permethrin. Six families showed cross resistance to both DDT and permethrin. Biochemical assays of F1 An. arabiensis families revealed comparatively high levels of monooxygenase (48%, n = 33, p<0.05), glutathione S-transferase (26%, n = 31, p<0.05) and general esterases activity compared to the reference colony. Insensitive acetylcholinesterase was detected in 23.5% (n = 33). The kdr analysis by PCR revealed presence of both East and West Africa mutation, but was not confirmed by sequencing. The significant elevation of various enzyme systems in the F1 progeny and detection of families showing cross resistance to permethrin and DDT is suggestive of existence of multiple resistances in An. arabiensis population from Gwave. This has serious implication on malaria control. Continued use of these insecticides is likely to further select resistant vectors. Use of mosaic insecticides or rotational use of insecticides is recommended