Genetic markers associated with the widespread insecticide resistance in malaria vector Anopheles funestus populations across Tanzania.

BACKGROUND: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. METHODS: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. RESULTS: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. CONCLUSIONS: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage.

Scaling artificial light at night and disease vector interactions into socio-ecological systems: a conceptual appraisal.

There is burgeoning interest in how artificial light at night (ALAN) interacts with disease vectors, particularly mosquitoes. ALAN can alter mosquito behaviour and biting propensity, and so must alter disease transfer rates. However, most studies to date have been laboratory-based, and it remains unclear how ALAN modulates disease vector risk. Here, we identify five priorities to assess how artificial light can influence disease vectors in socio-ecological systems. These are to (i) clarify the mechanistic role of artificial light on mosquitoes, (ii) determine how ALAN interacts with other drivers of global change to influence vector disease dynamics across species, (iii) determine how ALAN interacts with other vector suppression strategies, (iv) measure and quantify the impact of ALAN at scales relevant for vectors, and (v) overcome the political and social barriers in implementing it as a novel vector suppression strategy. These priorities must be addressed to evaluate the costs and benefits of employing appropriate ALAN regimes in complex socio-ecological systems if it is to reduce disease burdens, especially in the developing world. This article is part of the theme issue 'Light pollution in complex ecological systems'.

Advances in the genetic characterization of the malaria vector, Anopheles funestus, and implications for improved surveillance and control.

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

Bioactivity of select essential oil constituents against life stages of Anopheles arabiensis (Diptera: Culicidae).

Malaria is transmitted by infected female Anopheles mosquitoes, and An. arabiensis is a main malaria vector in arid African countries. Like other anophelines, its life cycle comprises of three aquatic stages; egg, larva, and pupa, followed by a free flying adult stage. Current vector control interventions using synthetic insecticides target these stages using adulticides or less commonly, larvicides. With escalating insecticide resistance against almost all conventional insecticides, identification of agents that simultaneously act at multiple stages of Anopheles life cycle presents a cost-effective opportunity. A further cost-effective approach would be the discovery of such insecticides from natural origin. Interestingly, essential oils present as potential sources of cost-effective and eco-friendly bioinsecticides. This study aimed to identify essential oil constituents (EOCs) with potential toxic effects against multiple stages of An. arabiensis life cycle. Five EOCs were assessed for inhibition of Anopheles egg hatching and ability to kill larvae, pupae and adult mosquitoes of An. arabiensis species. One of these EOCs, namely methyleugenol, exhibited potent Anopheles egg hatchability inhibition with an IC50 value of 0.51 ± 0.03 µM compared to propoxur (IC50: 5.13 ± 0.62 µM). Structure-activity relationship study revealed that methyleugenol and propoxur share a 1,2-dimethoxybenze moiety that may be responsible for the observed egg-hatchability inhibition. On the other hand, all five EOCs exhibited potent larvicidal activity with LC50 values less than 5 µM, with four of them; cis-nerolidol, trans-nerolidol, (-)-α-bisabolol, and farnesol, also possessing potent pupicidal effects (LC50 < 5 µM). Finally, all EOCs showed only moderate lethality against adult mosquitoes. This study reports for the first time, methyleugenol, (-)-α-bisabolol and farnesol as potent bioinsecticides against early life stages of An. arabiensis. This synchronized activity against Anopheles aquatic stages shows a prospect to integrate EOCs into existing adulticide-based vector control interventions.

The insecticidal activity of essential oil constituents against pyrethroid-resistant Anopheles funestus (Diptera: Culicidae).

Malaria vector control relies on the use of insecticides for indoor residual spraying and long-lasting bed nets. However, insecticide resistance to pyrethroids among others, has escalated. Anopheles funestus, one of the major African malaria vectors, has attained significant levels of resistance to pyrethroids. Overexpressed P450 monooxygenases have been previously identified in pyrethroid resistant An. funestus. The escalating resistance against conventional insecticides signals an urgent need for identification of novel insecticides. Essential oils have gained recognition as promising sources of alternative natural insecticides. This study investigated six essential oil constituents, farnesol, (-)-α-bisabolol, cis-nerolidol, trans-nerolidol, methyleugenol, santalol (α and ß isomers) and essential oil of sandalwood, for the adulticidal effects against pyrethroid-resistant An. funestus strain. The susceptibility against these terpenoids were evaluated on both pyrethroid-susceptible and resistant An. funestus. Furthermore, the presence of overexpressed monooxygenases in resistant An. funestus was confirmed. Results showed that both the pyrethroid-susceptible and resistant An. funestus were susceptible to three EOCs; cis-nerolidol, trans-nerolidol and methyleugenol. On the other hand, the pyrethroid-resistant An. funestus survived exposure to both farnesol and (-)-α-bisabolol. This study however does not show any direct association of the overexpressed Anopheles monooxygenases and the efficacy of farnesol and (-)-α-bisabolol. The enhanced activity of these terpenoids against resistant An. funestus that has been pre-exposed to a synergist, piperonyl butoxide, suggests their potential effectiveness in combination with monooxygenase inhibitors. This study proposes that cis-nerolidol, trans-nerolidol and methyleugenol are potential agents for further investigation as novel bioinsecticides against pyrethroid-resistant An. funestus strain.

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.

In vitro and in silico analysis of the Anopheles anticholinesterase activity of terpenoids.

Anopheles gambiae, An. coluzzii, An. arabiensis, and An. funestus are major vectors in high malaria endemic African regions. Various terpenoid classes form the main chemical constituent repository of essential oils, many of which have been shown to possess insecticidal effects against Anopheles species. The current study aimed to assess the bioactivity of terpenoids including four sesquiterpene alcohols, farnesol, (-)-α-bisabolol, cis-nerolidol, and trans-nerolidol; a phenylpropanoid, methyleugenol, and a monoterpene, (R)-(+)-limonene, using the larvicidal screening assay against the four Anopheles species. The mechanism of action was investigated through in vitro acetylcholinesterase inhibition assay and in silico molecular modelling. All six terpenoids showed potent larvicidal activity against the four Anopheles species. Insights into the mechanism of action revealed that the six terpenoids are strong AChE inhibitors against An. funestus and An. arabiensis, while there was a moderate inhibitory activity against An. gambiae AChE, but very weak activity against An. coluzzii. Interestingly, in the in silico study, farnesol established a favourable hydrogen bonding interaction with a conserved amino acid residue, Cys447, at the entrance to the active site gorge. While (-)-α-bisabolol and methyleugenol displayed a strong interaction with the catalytic Ser360 and adjacent amino acid residues; but sparing the mutable Gly280 residue that confers resistance to the current anticholinesterase insecticides. As a result, this study identified farnesol, (-)-α-bisabolol, and methyleugenol as selective bioinsecticidal agents with potent Anopheles AChE inhibition. These terpenoids present as natural compounds for further development as anticholinesterase bioinsecticides.

Relationships between biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes in south-eastern Tanzania.

BACKGROUND: Malaria transmission can be highly heterogeneous between and within localities, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the relationships between the biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes, which currently dominate malaria transmission in south-east Tanzania. The study also examined how such relationships may influence malaria transmission and control. METHODS: Female An. funestus were collected in houses located 50-100 m, 150-200 m or over 200 m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24 h-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity as a proxy for biological age. Adults emerging from larval collections in the same villages were tested against the same insecticides at 3-5, 8-11 or 17-20 days old. FINDINGS: Mosquitoes collected nearest to the aquatic habitats (50-100 m) had the lowest mortalities compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the insecticide-induced mortality assessed at both the diagnostic and multiplicative doses (1×, 5× and 10×) increased with mosquito age. The highest mortalities at 1× doses were observed among the oldest mosquitoes (17-20 days). At 10× doses, mortalities were 99% (permethrin) and 76% (deltamethrin) among 8-11 day-olds compared to 80% (permethrin) and 58% (deltamethrin) among 3-5 day-olds. Pre-exposure to PBO increased the potency of both pyrethroids. The proportion of parous females was highest among mosquitoes collected farthest from the habitats. CONCLUSION: In this specific setting, older An. funestus and those collected farthest from the aquatic habitats (near the centre of the village) were more susceptible to pyrethroids than the younger ones and those caught nearest to the habitats. These findings suggest that pyrethroid-based interventions may remain at least moderately effective despite widespread pyrethroid-resistance, by killing the older, less-resistant and potentially-infective mosquitoes. Further studies should investigate how and whether these observations could be exploited to optimize malaria control in different settings.

The in silico and in vitro analysis of donepezil derivatives for Anopheles acetylcholinesterase inhibition.

Current studies on Anopheles anticholinesterase insecticides are focusing on identifying agents with high selectivity towards Anopheles over mammalian targets. Acetylcholinesterase (AChE) from electric eel is often used as the bioequivalent enzyme to study ligands designed for activity and inhibition in human. In this study, previously identified derivatives of a potent AChE, donepezil, that have exhibited low activity on electric eel AChE were assessed for potential AChE-based larvicidal effects on four African malaria vectors; An. funestus, An. arabiensis, An. gambiae and An. coluzzii. This led to the identification of four larvicidal agents with a lead molecule, 1-benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 2 showing selectivity for An. arabiensis as a larvicidal AChE agent. Differential activities of this molecule on An. arabiensis and electric eel AChE targets were studied through molecular modelling. Homology modelling was used to generate a three-dimensional structure of the An. arabiensis AChE for this binding assay. The conformation of this molecule and corresponding interactions with the AChE catalytic site was markedly different between the two targets. Assessment of the differences between the AChE binding sites from electric eel, human and Anopheles revealed that the electric eel and human AChE proteins were very similar. In contrast, Anopheles AChE had a smaller cysteine residue in place of bulky phenylalanine group at the entrance to the catalytic site, and a smaller aspartic acid residue at the base of the active site gorge, in place of the bulky tyrosine residues. Results from this study suggest that this difference affects the ligand orientation and corresponding interactions at the catalytic site. The lead molecule 2 also formed more favourable interactions with An. arabiensis AChE model than other Anopheles AChE targets, possibly explaining the observed selectivity among other assessed Anopheles species. This study suggests that 1-benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 2 may be a lead compound for designing novel insecticides against Anopheles vectors with reduced toxic potential on humans.

Scientific Findings of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

For a decade, the Southern and Central Africa International Center of Excellence for Malaria Research has operated with local partners across study sites in Zambia and Zimbabwe that range from hypo- to holoendemic and vary ecologically and entomologically. The burden of malaria and the impact of control measures were assessed in longitudinal cohorts, cross-sectional surveys, passive and reactive case detection, and other observational designs that incorporated multidisciplinary scientific approaches: classical epidemiology, geospatial science, serosurveillance, parasite and mosquito genetics, and vector bionomics. Findings to date have helped elaborate the patterns and possible causes of sustained low-to-moderate transmission in southern Zambia and eastern Zimbabwe and recalcitrant high transmission and fatality in northern Zambia. Cryptic and novel mosquito vectors, asymptomatic parasite reservoirs in older children, residual parasitemia and gametocytemia after treatment, indoor residual spraying timed dyssynchronously to vector abundance, and stockouts of essential malaria commodities, all in the context of intractable rural poverty, appear to explain the persistent malaria burden despite current interventions. Ongoing studies of high-resolution transmission chains, parasite population structures, long-term malaria periodicity, and molecular entomology are further helping to lay new avenues for malaria control in southern and central Africa and similar settings.

Policy Implications of the Southern and Central Africa International Center of Excellence for Malaria Research: Ten Years of Malaria Control Impact Assessments in Hypo-, Meso-, and Holoendemic Transmission Zones in Zambia and Zimbabwe.

The International Centers of Excellence for Malaria Research (ICEMR) were established by the National Institute of Allergy and Infectious Diseases more than a decade ago to provide multidisciplinary research support to malaria control programs worldwide, operating in endemic areas and contributing technology, expertise, and ultimately policy guidance for malaria control and elimination. The Southern and Central Africa ICEMR has conducted research across three main sites in Zambia and Zimbabwe that differ in ecology, entomology, transmission intensity, and control strategies. Scientific findings led to new policies and action by the national malaria control programs and their partners in the selection of methods, materials, timing, and locations of case management and vector control. Malaria risk maps and predictive models of case detection furnished by the ICEMR informed malaria elimination programming in southern Zambia, and time series analyses of entomological and parasitological data motivated several major changes to indoor residual spray campaigns in northern Zambia. Along the Zimbabwe-Mozambique border, temporal and geospatial data are currently informing investigations into a recent resurgence of malaria. Other ICEMR findings pertaining to parasite and mosquito genetics, human behavior, and clinical epidemiology have similarly yielded immediate and long-term policy implications at each of the sites, often with generalizable conclusions. The ICEMR programs thereby provide rigorous scientific investigations and analyses to national control and elimination programs, without which the impediments to malaria control and their potential solutions would remain understudied.

The realized efficacy of indoor residual spraying campaigns falls quickly below the recommended WHO threshold when coverage, pace of spraying and residual efficacy on different wall types are considered.

Indoor residual spraying (IRS) has been and remains an important malaria control intervention in southern Mozambique, South Africa and Eswatini. A better understanding of the effectiveness of IRS campaigns is critical to guide future elimination efforts. We analyze the three IRS campaigns conducted during a malaria elimination demonstration project in southern Mozambique, the "Magude project", and propose a new method to calculate the efficacy of IRS campaigns adjusting for IRS coverage, pace of house spraying and IRS residual efficacy on different wall types. Anopheles funestus sensu lato (s.l.) and An. gambiae s.l. were susceptible to pirimiphos-methyl and DDT. Anopheles funestus s.l. was resistant to pyrethroids, with 24h post-exposure mortality being lower for An. funestus sensu stricto (s.s.) than for An. parensis (collected indoors). The percentage of structures sprayed was above 90% and percentage of people covered above 86% in all three IRS campaigns. The percentage of households sprayed was above 83% in 2015 and 2016, but not assessed in 2017. Mosquito mortality 24h post-exposure stayed above 80% for 196 days after the 2016 IRS campaign and 222 days after the 2017 campaign and was 1.5 months longer on mud walls than on cement walls. This was extended by up to two months when 120h post-exposure mortality was considered. The district-level realized IRS efficacy was 113 days after the 2016 campaign. While the coverage of IRS campaigns in Magude were high, IRS protection did not remain optimal for the entire high malaria transmissions season. The use of a longer-lasting IRS product could have further supported the interruption of malaria transmission in the district. To better estimate the protection afforded by IRS campaigns, National Malaria Control Programs and partners are encouraged to adjust the calculation of IRS efficacy for IRS coverage, pace of house spraying during the campaign and IRS efficacy on different wall types combined with wall type distribution in the sprayed area.

Potential of Essential Oil-Based Anticholinesterase Insecticides against Anopheles Vectors: A Review.

The insect nervous system is critical for its functional integrity. The cholinergic system, of which acetylcholinesterase (AChE) is a key enzyme, is essential to the Anopheles (consisting of major malaria vector species) nervous system. Furthermore, the nervous system is also the primary target site for insecticides used in malaria vector control programs. Insecticides, incorporated in insecticide-treated nets and used for indoor residual spraying, are a core intervention employed in malaria vector control. However, Anopheles resistance against these insecticides has grown rapidly. Due to this major setback, novel agents with potential activity against resistant Anopheles and/or capacity to overcome resistance against current WHO-approved insecticides are urgently needed. The essential oils have the potential to be natural sources of novel insecticides with potential to inhibit the Anopheles AChE target. In the current review, the scientific evidence highlights the ability of essential oils and specific essential oil constituents to serve as anticholinesterase insecticides. For this reason, the published data from scientific databases on the essential oils and essential oil constituents on anticholinesterase, ovicidal, larvicidal, pupicidal and adulticidal activities were analyzed. The identification of major constituents in active essential oils and their possible influence on the biological activity have also been critically evaluated. Furthermore, the toxicity to mammals as well as potential activity against the mammalian AChE target has also been reviewed. The importance of identifying novel potent insecticides from essential oils has been discussed, in relation to human safety and cost-effectiveness. Finally, the critical insights from this review can be used to inform future researchers towards potent and safe anticholinesterase insecticides for the management of Anopheles malaria vectors.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

The mosquito vectors that sustained malaria transmission during the Magude project despite the combined deployment of indoor residual spraying, insecticide-treated nets and mass-drug administration.

The "Magude project" aimed but failed to interrupt local malaria transmission in Magude district, southern Mozambique, by using a comprehensive package of interventions, including indoor residual spraying (IRS), pyrethroid-only long-lasting insecticide treated nets (LLINs) and mass-drug administration (MDA). Here we present detailed information on the vector species that sustained malaria transmission, their association with malaria incidence and behaviors, and their amenability to the implemented control interventions. Mosquitoes were collected monthly between May 2015 and October 2017 in six sentinel sites in Magude district, using CDC light traps both indoors and outdoors. Anopheles arabiensis was the main vector during the project, while An. funestus s.s., An. merus, An. parensis and An. squamosus likely played a secondary role. The latter two species have never previously been found positive for Plasmodium falciparum in southern Mozambique. The intervention package successfully reduced vector sporozoite rates in all species throughout the project. IRS was effective in controlling An. funestus s.s. and An. parensis, which virtually disappeared after its first implementation, but less effective at controlling An. arabiensis. Despite suboptimal use, LLINs likely provided significant protection against An. arabiensis and An. merus that sought their host largely indoors when people where in bed. Adding IRS on top of LLINs and MDA likely added value to the control of malaria vectors during the Magude project. Future malaria elimination attempts in the area could benefit from i) increasing the use of LLINs, ii) using longer-lasting IRS products to counteract the increase in vector densities observed towards the end of the high transmission season, and iii) a higher coverage with MDA to reduce the likelihood of human infection. However, additional interventions targeting vectors that survive IRS and LLINs by biting outdoors or indoors before people go to bed, will be likely needed to achieve local malaria elimination.

Expanded geographic distribution and host preference of Anopheles gibbinsi (Anopheles species 6) in northern Zambia.

BACKGROUND: Nchelenge District in northern Zambia suffers from holoendemic malaria transmission despite a decade of yearly indoor residual spraying (IRS) and insecticide-treated net (ITN) distributions. One hypothesis for this lack of impact is that some vectors in the area may forage in the early evening or outdoors. Anopheles gibbinsi specimens were identified in early evening mosquito collections performed in this study area, and further insight was gleaned into this taxon, including characterizing its genetic identity, feeding preferences, and potential role as a malaria vector. METHODS: Mosquitoes were collected in July and August 2019 by CDC light traps in Nchelenge District in indoor sitting rooms, outdoor gathering spaces, and animal pens from 16:00-22:00. Host detection by PCR, COI and ITS2 PCR, and circumsporozoite (CSP) ELISA were performed on all samples morphologically identified as An. gibbinsi, and a subset of specimens were selected for COI and ITS2 sequencing. To determine risk factors for increased abundance of An. gibbinsi, a negative binomial generalized linear mixed-effects model was performed with household-level variables of interest. RESULTS: Comparison of COI and ITS2 An. gibbinsi reference sequences to the NCBI database revealed > 99% identity to "Anopheles sp. 6" from Kenya. More than 97% of specimens were morphologically and molecularly consistent with An. gibbinsi. Specimens were primarily collected in animal pen traps (59.2%), followed by traps outdoors near where humans gather (24.3%), and traps set indoors (16.5%). Host DNA detection revealed a high propensity for goats, but 5% of specimens with detected host DNA had fed on humans. No specimens were positive for Plasmodium falciparum sporozoites. Animal pens and inland households > 3 km from Lake Mweru were both associated with increased An. gibbinsi abundance. CONCLUSIONS: This is the first report of An. gibbinsi in Nchelenge District, Zambia. This study provided a species identity for unknown "An. sp. 6" in the NCBI database, which has been implicated in malaria transmission in Kenya. Composite data suggest that this species is largely zoophilic and exophilic, but comes into contact with humans and the malaria parasites they carry. This species should continue to be monitored in Zambia and neighbouring countries as a potential malaria vector.

To spray or target mosquitoes another way: focused entomological intelligence guides the implementation of indoor residual spraying in southern Mozambique.

BACKGROUND: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics. METHODS: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps. RESULTS: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled. CONCLUSION: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary malaria vector suggesting that IRS can reduce malaria transmission, but the presence of other vector species both indoors and outdoors suggests that alternative vector control interventions that target these gaps in protection may increase the impact of vector control in southern Mozambique.

To spray or target mosquitoes another way: focused entomological intelligence guides the implementation of indoor residual spraying in southern Mozambique

Background: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics. Methods: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps. Results: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled. Conclusion: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary...

Intensity of insecticide resistance in the major malaria vector Anopheles funestus from Chikwawa, rural Southern Malawi.

BACKGROUND: Malaria vector control using insecticide-based approaches has proven to be an effective strategy. However, widespread insecticide resistance among malaria vector populations across sub-Saharan Africa threatens to derail control efforts. This study was conducted in Chikwawa district, an area in rural southern Malawi characterised by persistent malaria transmission and reports of insecticide resistance in the local mosquito population. The aim of the was to characterise the intensity of insecticide resistance within a population of Anopheles funestus sensu lato (s.l.), a major vector of malaria in this district. METHODS: Live adult females belonging to the An. funestus group were collected from households by indoor aspiration. The CDC bottle assay was used for phenotypic quantification of resistance to deltamethrin, permethrin and alpha-cypermethrin at 1×, 2.5×, 5× and 10× the recommended diagnostic dose for each of these insecticides. WHO tube assays were used to determine susceptibility to bendiocarb, dichlorodiphenyltrichloroethane (DDT) and pirimiphos-methyl insecticides at diagnostic concentrations. RESULTS: Anopheles funestus s.l. exposed to 10× the recommended diagnostic dose was highly resistant to alpha-cypermethrin (mortality 95.4%); in contrast, mortality was 100% when exposed to both deltamethrin and permethrin at the same dose. Despite showing susceptibility to deltamethrin and permethrin at the 10× concentration, mortality at the 5× concentration was 96.7% and 97.1%, respectively, indicating moderate resistance to these two insecticides. WHO susceptibility assays indicated strong resistance against bendiocarb (mortality 33.8%, n = 93), whereas there was full susceptibility to DDT (mortality 98.9%, n = 103) and pirimiphos-methyl (mortality 100%, n = 103). CONCLUSIONS: Strategies for managing resistance to insecticides, particularly against pyrethroids, must be urgently implemented to maintain the effectiveness of insecticide-based vector control interventions in the area. Such strategies include the wide-scale introduction of third-generation synergist insecticide-treated bed nets (ITNs) and next-generation dual active ingredient ITNs. The use of effective non-pyrethroids, such as pirimiphos-methyl, clothianidin and potentially DDT, could provide a window of opportunity for indoor residual spraying across the district. This strategy would support the current Malawi Insecticide Resistance Management Plan which aims at rotating insecticides to minimise selection pressure and slow down the evolution of resistance to approved insecticides. These actions will help to prevent malaria vector control failure and improve progress towards malaria elimination.