A review of historical trends in Anopheles gambiae Giles (Diptera: Culicidae) complex composition, collection trends and environmental effects from 2009 to 2021 in Mpumalanga province, South Africa.

South Africa is a frontline country for malaria elimination in the southern African region. It has three malaria-endemic provinces, each with its own transmission pattern. The elimination of malaria depends, in part, on controlling and/or eliminating vectors responsible for transmission. Sustained entomological surveillance is an important factor to consider when shifting from a control to elimination framework. The Ehlanzeni district in Mpumalanga province is a key entomological sentinel surveillance area. It is one of the malaria-endemic districts in South Africa with higher rates of malaria incidences. As such, entomological data about the Anopheles gambiae Giles (Diptera: Culicidae) complex have been collected in this province over a substantial period. These data are stored in a pre-existing institutional database. An analysis of the trends that can be observed from this database has not been performed before. This retrospective (longitudinal) analysis provides a summary of the An. gambiae complex vector composition in this region from 2009 to 2021. Routine surveillance data were correlated with climatic data (obtained from the NASA LaRC POWER project database) for the same period to assess the role of climatic factors in vector dynamics. This review also identifies a number of limitations in the data collection process across the sampling period and provides recommendations on how to strengthen the database going forward. The most abundant member of the An. gambiae complex since 2009 in the province was An. merus Dönitz followed by An. arabiensis Patton. Collection methods used showed that human landing catches were successful for collecting An. arabiensis, while pit traps were the most effective in collecting An. merus and An. quadriannulatus Theobald. The latter two species were mainly collected in spring, whereas An. arabiensis abundance was larger during autumn collections. Vector abundance was not significantly correlated with annual climatic data. The information gained from this database provides insights into the vector dynamics of the Ehlanzeni district of the Mpumalanga province.

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.

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.

Staggered larval time-to-hatch and insecticide resistance in the major malaria vector Anopheles gambiae S form.

BACKGROUND: Anopheles gambiae is a major vector of malaria in the West African region. Resistance to multiple insecticides has been recorded in An. gambiae S form in the Ahafo region of Ghana. A laboratory population (GAH) established using wild material from this locality has enabled a mechanistic characterization of each resistance phenotype as well as an analysis of another adaptive characteristic - staggered larval time-to-hatch. METHODS: Individual egg batches obtained from wild caught females collected from Ghana and the Republic of the Congo were monitored for staggered larval time-to-hatch. In addition, early and late larval time-to-hatch sub-colonies were selected from GAH. These selected sub-colonies were cross-mated and their hybrid progeny were subsequently intercrossed and back-crossed to the parental strains. The insecticide susceptibilities of the GAH base colony and the time-to-hatch selected sub-colonies were quantified for four insecticide classes using insecticide bioassays. Resistance phenotypes were mechanistically characterized using insecticide-synergist bioassays and diagnostic molecular assays for known reduced target-site sensitivity mutations. RESULTS: Anopheles gambiae GAH showed varying levels of resistance to all insecticide classes. Metabolic detoxification and reduced target-site sensitivity mechanisms were implicated. Most wild-caught families showed staggered larval time-to-hatch. However, some families were either exclusively early hatching or late hatching. Most GAH larvae hatched early but many egg batches contained a proportion of late hatching larvae. Crosses between the time-to-hatch selected sub-colonies yielded ambiguous results that did not fit any hypothetical models based on single-locus Mendelian inheritance. There was significant variation in the expression of insecticide resistance between the time-to-hatch phenotypes. CONCLUSIONS: An adaptive response to the presence of multiple insecticide classes necessarily involves the development of multiple resistance mechanisms whose effectiveness may be enhanced by intra-population variation in the expression of resistance phenotypes. The variation in the expression of insecticide resistance in association with selection for larval time-to-hatch may induce this kind of enhanced adaptive plasticity as a consequence of pleiotropy, whereby mosquitoes are able to complete their aquatic life stages in a variable breeding environment using staggered larval time-to-hatch, giving rise to an adult population with enhanced variation in the expression of insecticide resistance.

Embryonic development and rates of metabolic activity in early and late hatching eggs of the major malaria vector Anopheles gambiae.

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO2 output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.

Insecticide resistance in malaria vector mosquitoes at four localities in Ghana, West Africa.

BACKGROUND: Malaria vector control programmes that rely on insecticide-based interventions such as indoor house spraying with residual insecticides or insecticide treated bed nets, need to base their decision-making process on sound baseline data. More and more commercial entities in Africa, such as mining companies, are realising the value to staff productivity of controlling malaria transmission in their areas of operation.This paper presents baseline entomological data obtained during surveys conducted for four mining operations in Ghana, West Africa. RESULTS: The vast majority of the samples were identified as Anopheles gambiae S form with only a few M form specimens being identified from Tarkwa. Plasmodium falciparum infection rates ranged from 4.5 to 8.6% in An. gambiae and 1.81 to 8.06% in An. funestus. High survival rates on standard WHO bioassay tests were recorded for all insecticide classes except the organophosphates that showed reasonable mortality at all locations (i.e. >90%). The West African kdr mutation was detected and showed high frequencies in all populations. CONCLUSIONS: The data highlight the complexity of the situation prevailing in southern Ghana and the challenges facing the malaria vector control programmes in this region. Vector control programmes in Ghana need to carefully consider the resistance profiles of the local mosquito populations in order to base their resistance management strategies on sound scientific data.

Multiple insecticide resistance in Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo.

Successful implementation of an integrated vector control program will rely on availability of accurate vector information in the specific location. However, such information can be limited in some countries. The aim of this study was to obtain baseline vector information from Pointe Noire on the Congo coast (Republic of the Congo). Field sampling was conducted during April 2009 in the village of Boutoto and its surrounds, close to the city of Pointe Noire. Anopheles gambiae sensu lato mosquitoes were collected resting indoors. Samples were analyzed for insecticide susceptibility, species identification, and Plasmodium sporozoite infection. Molecular and biochemical assays were conducted to characterize insecticide resistance mechanisms. The malaria vector A. gambiae S-form was the only mosquito species identified, and it had a high Plasmodium falciparum infection rate (9.6%). Multiple insecticide resistance was detected in this population with full susceptibility to only one insecticide class, the organophosphates. Dieldrin and DDT resistance was mainly attributed to target-site resistance (the Rdl and L1014F/L1014S kdr mutations respectively), whereas pyrethroid resistance was mainly attributed to P450 metabolic enzyme-mediated detoxification in addition to kdr. The role of various insecticide resistance mechanisms revealed a complex association between metabolic detoxification and reduced target-site sensitivity.