Potential Mosquito Vectors for Shuni Virus, South Africa, 2014-2018.

Shuni virus is associated with neurologic and febrile illness in animals and humans. To determine potential vectors, we collected mosquitoes in South Africa and detected the virus in species of the genera Mansonia, Culex, Aedes, and Anopheles. These mosquitoes may be associated with Shuni virus outbreaks in Africa and emergence in other regions.

Member species of the Anopheles gambiae complex can be misidentified as Anopheles leesoni.

BACKGROUND: Accurate Anopheles species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the Anopheles funestus group multiplex PCR assay, it was noted that Anopheles arabiensis can be misidentified as Anopheles leesoni, a zoophilic member of the An. funestus group. The aim of this project was, therefore, to ascertain whether other members of the Anopheles gambiae complex can also be misidentified as An. leesoni when using the standard An. funestus multiplex PCR. METHODS: The An. funestus multiplex PCR was used to amplify DNA from An. gambiae complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and An. leesoni species-specific primers (LEES), were sequence analysed. Additionally, An. leesoni DNA was processed through the diagnostic An. gambiae multiplex PCR to determine if this species can be misidentified as a member of the An. gambiae complex. RESULTS: Laboratory-colonized as well as field-collected samples of An. arabiensis, An. gambiae, Anopheles merus, Anopheles quadriannulatus, Anopheles coluzzii as well as Anopheles moucheti produced an amplicon of similar size to that of An. leesoni when using an An. funestus multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the An. gambiae complex and An. moucheti. The reverse was not true, i.e. the An. gambiae multiplex PCR does not amplify DNA from An. leesoni. CONCLUSION: This investigation shows that An. arabiensis, An. gambiae, An. merus, An. quadriannulatus, An. coluzzii and An. moucheti can be misidentified as An. leesoni when using An. funestus multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as An. leesoni, the An. gambiae multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

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.

Anopheles parensis contributes to residual malaria transmission in South Africa.

BACKGROUND: Understanding the contribution of outdoor-resting Anopheles mosquitoes to residual malaria transmission is important in terms of scaling up vector control towards malaria elimination in South Africa. The aim of this project was to assess the potential role of Anopheles parensis and other Anopheles species in residual malaria transmission, using sentinel surveillance sites in the uMkhanyakude District of northern KwaZulu-Natal Province. METHODS: Monthly vector surveillance was conducted at the sentinel sites from January 2017 to May 2018. Outdoor-placed clay pot resting traps were used to collect male and female adult Anopheles mosquitoes. All Anopheles gambiae complex and Anopheles funestus group specimens collected were identified to species and all females were screened for Plasmodium falciparum circumsporozoite protein (CSP) by enzyme-linked immunosorbent assay (ELISA). Samples showing infectivity for P. falciparum were further verified by a nested PCR and subsequent DNA sequence analysis. RESULTS: From a sample of 491 anophelines, Anopheles arabiensis (n = 228) and An. parensis (n = 194) were the most abundant. Other species collected included Anopheles merus (n =11), Anopheles quadriannulatus (n = 10), Anopheles leesoni (n = 29), Anopheles rivulorum (n =18), and Anopheles vaneedeni (n =1). Of the 317 female specimens screened for P. falciparum CSP, one Anopheles arabiensis and one An. parensis showed positive by ELISA and Plasmodium nested PCR. For the An. parensis specimen, confirmation of its species identity was based on sequence analysis of the ITS2 region, and the presence of P. falciparum DNA was further confirmed by sequence analysis. CONCLUSIONS: Anopheles parensis is a potential vector of malaria in South Africa although its contribution to transmission is likely to be minimal at best owing to its strong zoophilic tendency. By contrast, An. arabiensis is a major vector that is primarily responsible for the bulk of residual malaria transmission in South Africa. As all recently collected sporozoite-positive Anopheles mosquitoes were found in outdoor-placed resting traps, it is necessary to introduce interventions that can be used to control outdoor-resting vector populations while maintaining the efficacy of South Africa's indoor house spraying operations.

Electrostatic coating enhances bioavailability of insecticides and breaks pyrethroid resistance in mosquitoes.

Insecticide resistance poses a significant and increasing threat to the control of malaria and other mosquito-borne diseases. We present a novel method of insecticide application based on netting treated with an electrostatic coating that binds insecticidal particles through polarity. Electrostatic netting can hold small amounts of insecticides effectively and results in enhanced bioavailability upon contact by the insect. Six pyrethroid-resistant Anopheles mosquito strains from across Africa were exposed to similar concentrations of deltamethrin on electrostatic netting or a standard long-lasting deltamethrin-coated bednet (PermaNet 2.0). Standard WHO exposure bioassays showed that electrostatic netting induced significantly higher mortality rates than the PermaNet, thereby effectively breaking mosquito resistance. Electrostatic netting also induced high mortality in resistant mosquito strains when a 15-fold lower dose of deltamethrin was applied and when the exposure time was reduced to only 5 s. Because different types of particles adhere to electrostatic netting, it is also possible to apply nonpyrethroid insecticides. Three insecticide classes were effective against strains of Aedes and Culex mosquitoes, demonstrating that electrostatic netting can be used to deploy a wide range of active insecticides against all major groups of disease-transmitting mosquitoes. Promising applications include the use of electrostatic coating on walls or eave curtains and in trapping/contamination devices. We conclude that application of electrostatically adhered particles boosts the efficacy of WHO-recommended insecticides even against resistant mosquitoes. This innovative technique has potential to support the use of unconventional insecticide classes or combinations thereof, potentially offering a significant step forward in managing insecticide resistance in vector-control operations.

The effect of elevated temperatures on the life history and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

BACKGROUND: Temperature plays a crucial role in the life history of insects. Recent climate change research has highlighted the importance of elevated temperature on malaria vector distribution. This study aims to examine the role of elevated temperatures on epidemiologically important life-history traits in the major malaria vector, Anopheles arabiensis. Specifically, the differential effects of temperature on insecticide-resistant and susceptible strains were examined. METHODS: Two laboratory strains of A. arabiensis, the insecticide-susceptible SENN and the insecticide-resistant SENN DDT strains, were used to examine the effect of elevated temperatures on larval development and adult longevity. The effects of various elevated temperatures on insecticide resistance phenotypes were also examined and the biochemical basis of the changes in insecticide resistance phenotype was assessed. RESULTS: SENN and SENN DDT larvae developed at similar rates at elevated temperatures. SENN DDT adult survivorship did not vary between control and elevated temperatures, while the longevity of SENN adults at constantly elevated temperatures was significantly reduced. SENN DDT adults lived significantly longer than SENN at constantly elevated temperatures. Elevated rearing temperatures, as well as a short-term exposure to 37 and 39 °C as adults, augmented pyrethroid resistance in adult SENN DDT, and increased pyrethroid tolerance in SENN. Detoxification enzyme activity was not implicated in this phenotypic effect. Quercertin-induced synergism of inducible heat shock proteins negated this temperature-mediated augmentation of pyrethroid resistance. CONCLUSION: Insecticide-resistant A. arabiensis live longer than their susceptible counterparts at elevated temperatures. Exposure to heat shock augments pyrethroid resistance in both resistant and susceptible strains. This response is potentially mediated by inducible heat shock proteins.

Reviewing South Africa's malaria elimination strategy (2012-2018): progress, challenges and priorities.

BACKGROUND: With a sustained national malaria incidence of fewer than one case per 1000 population at risk, in 2012 South Africa officially transitioned from controlling malaria to the ambitious goal of eliminating malaria within its borders by 2018. This review assesses the progress made in the 3 years since programme re-orientation while highlighting challenges and suggesting priorities for moving the malaria programme towards elimination. METHODS: National malaria case data and annual spray coverage data from 2010 until 2014 were assessed for trends. Information on surveillance, monitoring and evaluation systems, human and infrastructure needs and community malaria knowledge was sourced from the national programme mid-term review. RESULTS: Malaria cases increased markedly from 6811 in 2013 to 11,711 in 2014, with Mpumalanga and Limpopo provinces most affected. Enhanced local transmission appeared to drive malaria transmission in Limpopo Province, while imported malaria cases accounted for the majority of cases reported in Mpumalanga Province. Despite these increases only Vhembe and Mopani districts in Limpopo Province reported malaria incidences more than one case per 1000 population at risk by 2014. Over the review period annual spray coverage did not reach the recommended target of 90 % coverage, with information gaps identified in parasite prevalence, artemether-lumefantrine therapeutic utilization, asymptomatic/sub-patent carriage, drug efficacy, vector distribution and insecticide resistance. CONCLUSIONS: Although South Africa has made steady progress since adopting an elimination agenda, a number of challenges have been identified. The heterogeneity of malaria transmission suggests interventions in Vhembe and Mopani districts should focus on control, while in KwaZulu-Natal Province eliminating transmission foci should be prioritized. Cross-border initiatives with neighbouring countries should be established/strengthened as a matter of urgency since malaria importation poses a real threat to the country's elimination efforts. It is also critical that provincial programmes are adequately resourced to effectively conduct the necessary targeted elimination activities, informed by current vector/parasite distribution and resistance data. More sensitive methods to detect sub-patent infections, primaquine as a transmission-blocking drug, and alternative vector control methods need to be investigated. Knowledge gaps among malaria health workers and affected communities should be identified and addressed.

The neonicotinoid imidacloprid, and the pyrethroid deltamethrin, are antagonists of the insect Rdl GABA receptor.

A mutation in the second transmembrane domain of the GABA receptor subunit, Rdl, is associated with resistance to insecticides such as dieldrin and fipronil. Molecular cloning of Rdl cDNA from a strain of the malaria mosquito, Anopheles gambiae, which is highly resistant to dieldrin revealed this mutation (A296G) as well as another mutation in the third transmembrane domain (T345M). Wild-type, A296G, T345M and A296G + T345M homomultimeric Rdl were expressed in Xenopus laevis oocytes and their sensitivities to fipronil, deltamethrin, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), imidacloprid and spinosad were measured using two-electrode voltage-clamp electrophysiology. Spinosad and DDT had no agonist or antagonist actions on Rdl. However, fipronil, deltamethrin and imidacloprid decreased GABA-evoked currents. These antagonistic actions were either reduced or abolished with the A296G and the A296G + T345M mutations while T345M alone appeared to have no significant effect. In conclusion, this study identifies another mutation in the mosquito Rdl that is associated with insecticide resistance. While T345M itself does not affect insecticide sensitivity, it may serve to offset the structural impact of A296G. The present study also highlights Rdl as a potential secondary target for neonicotinoids and pyrethroids. We show for the first time that deltamethrin (a pyrethroid insecticide) and imidacloprid (a neonicotinoid insecticide) act directly on the insect GABA receptor, Rdl. Our findings highlight Rdl as a potential secondary target of pyrethroids and neonicotinoids mutations in which may contribute to resistance to these widely used insecticides.

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 effect of larval nutritional deprivation on the life history and DDT resistance phenotype in laboratory strains of the malaria vector Anopheles arabiensis.

BACKGROUND: Anopheles arabiensis is a major malaria vector in Africa. It thrives in agricultural areas and has been associated with increased malaria incidence in areas under rice and maize cultivation. This effect may be due to increased adult size and abundance as a consequence of optimal larval nutrition. The aim of this study was to examine the effect of larval nutrition on the life history and expression of insecticide resistance in adults of laboratory reared An. arabiensis. METHODS: Larvae drawn from an insecticide susceptible An. arabiensis strain (SENN) as well as a DDT-resistant strain (SENN-DDT) were subjected to three fasting regimes: 1 mg of food per larva offered once per day, once every second day and once every third day. Control cohorts included larvae offered 1 mg food thrice per day. The rate of larval development was compared between matched cohorts from each strain as well as between fasted larvae and their respective controls. The expression of DDT resistance/tolerance in adults was compared between the starved cohorts and their controls by strain. Factors potentially affecting variation in DDT resistance/tolerance were examined including: adult body size (wing length), knock-down resistance (kdr) status and levels of detoxification enzyme activity. RESULTS AND CONCLUSION: Anopheles arabiensis larval development is prolonged by nutrient deprivation and adults that eclose from starved larvae are smaller and less tolerant to DDT intoxication. This effect on DDT tolerance in adults is also associated with reduced detoxification enzyme activity. Conversely, well fed larvae develop comparatively quickly into large, more DDT tolerant (SENN) or resistant (SENN-DDT) adults. This is important in those instances where cereal farming is associated with increased An. arabiensis transmitted malaria incidence, because large adult females with high teneral reserves and decreased susceptibility to insecticide intoxication may also prove to be more efficient malaria vectors. In general, larval nutrient deprivation in An. arabiensis has important implications for subsequent adults in terms of their size and relative insecticide susceptibility, which may in turn impact on their malaria vector capacity in areas where insecticide based control measures are in place.

Alphaviruses Detected in Mosquitoes in the North-Eastern Regions of South Africa, 2014 to 2018.

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Fungal infection counters insecticide resistance in African malaria mosquitoes.

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies.

Malaria Vector Surveillance and Control in an Elimination Setting in South Africa.

South Africa's malaria elimination plans are aligned to the World Health Organization's aim for a malaria-free world and include specific objectives within a specified time frame. These are proving difficult to achieve owing to the sporadic nature of locally acquired malaria in some affected districts, while other districts that were endemic for the disease are either malaria-free or very close to that goal. The WHO also specifies that continued measures to prevent the re-establishment of transmission are required in areas where elimination has been achieved. These measures include routine malaria vector surveillance in endemic districts that are free of malaria to assess receptivity and risk of reintroduction, which may prove difficult to justify in the face of competing public health priorities and limited resources. These issues are discussed here within the framework of vector surveillance and control and include recommendations on how they can be addressed going forward.

The effect of blood feeding on insecticide resistance intensity and adult longevity in the major malaria vector Anopheles funestus (Diptera: Culicidae).

Insecticide-based vector control is key to the reduction and elimination of malaria. Although insecticide resistance is common in malaria vector populations, the operational implications are often unclear. High intensity pyrethroid resistance in the major malaria vector Anopheles funestus has been linked to control failure in Southern Africa. The aim of this study was to assess linkages between mosquito age, blood feeding and the intensity of pyrethroid resistance in two An. funestus laboratory strains that originate from southern Mozambique, namely the moderately pyrethroid resistant FUMOZ and the highly resistant FUMOZ-R. Resistance tended to decline with age. This effect was significantly mitigated by blood feeding and was most apparent in cohorts that received multiple blood meals. In the absence of insecticide exposure, blood feeding tended to increase longevity of An. funestus females and, following insecticide exposure, enhanced their levels of deltamethrin resistance, even in older age groups. These effects were more marked in FUMOZ-R compared to FUMOZ. In terms of programmatic decision-making, these data suggest that it would be useful to assess the level and intensity of resistance in older female cohorts wherever possible, notwithstanding the standard protocols for resistance testing using age-standardised samples.

Characterisation of the epigenetic architecture of the major malaria vector Anopheles arabiensis (Diptera: Culicidae) after treatment with epigenetic modulators and heavy metals.

Anopheles arabiensis (a member of the An. gambiae species complex) is a major vector of malaria in sub-Saharan Africa. Despite its disease vector status, there is currently a paucity of epigenetic information for this species. The aim this study was therefore to analyse global epigenetic markers and their response to metal exposure in insecticide susceptible and resistant laboratory strains of An. arabiensis. This was done using commercially available epigenetic marker quantification kits. In order to validate the efficacy of the kits, several kits were assessed to determine whether changes induced by known epigenetic modulators were detectable using these platforms. The efficacy of the dosages used were determined by examining the effect of the dosages used on insecticide resistant phenotypes. Upon confirmation that the dosages used were sufficient to induce a phenotypic change, the effect on epigenetic markers was assessed. Commercial kits were used to quantify 5-methylcysteine (5-mC) and 5-hydroxymethylcysteine (5-hmC) methylation in DNA, m6A methylation in mRNA as well as Histone Acetyl Transferase (HAT) activity. There was a marked difference in the phenotypic response in adult mosquitoes of the insecticide susceptible strain compared to that of its' resistant counterpart. For males and females of the resistant strain, exposure to nucleic acid modifying drugs typically increased their tolerance to insecticides. The patterns of changes in 5-mC methylation by epigenetic modulators was congruent with previous studies which quantified by mass spectrometry. The two strains differed in methylation patterns under control conditions and responded differentially to larval metal exposure. In the resistant strain, which previously was demonstrated to show increased detoxification enzyme activity and insecticide tolerance after the same treatment, the potential increase in transcriptional activity appeared to be modulated by reduced methylation and increased HAT activity. This study suggests that the commercial epigenetic quantification kits can be used to characterise phenotypic changes in An. arabiensis, and also shows that epigenetic regulation of the response to metal exposure is regulated at the DNA as opposed to the RNA level.

Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa.

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

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.

The infectivity of the entomopathogenic fungus Beauveria bassiana to insecticide-resistant and susceptible Anopheles arabiensis mosquitoes at two different temperatures.

BACKGROUND: Control of the major African malaria vector species continues to rely extensively on the application of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is undermining the sustainability of these control strategies. Alternatives to the currently available conventional chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such possibility. However, one of the challenges to the approach is the potential influence of varied environmental conditions and target species that could affect the efficacy of a biological 'active ingredient'. An initial investigation into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different laboratory temperature regimes. METHODS: Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 +/- 1 degrees C or 25 +/- 2 degrees C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and temperature on mortality rates following fungus infection. RESULTS: Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana. All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature treatments with virulence significantly higher at 25 degrees C than 21 degrees C. Even so, because malaria parasite development is also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal infection under the cooler conditions would still be high. CONCLUSIONS: These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study to optimize selection of isolates, dose and use strategy in different eco-epidemiological settings.