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Several trials and reviews have outlined the potential role of larviciding for malaria control in sub-Saharan Africa (SSA) to supplement the core indoor insecticide-based interventions. It has been argued that widespread use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) interventions in many parts of Africa result in many new areas with low and focal malaria transmission that can be targeted with larvicides. As some countries in SSA are making good progress in malaria control, larval source management, particularly with bacterial larvicides, could be included in the list of viable options to maintain the gains achieved while paving the way to malaria elimination. We conducted a review of published literature that investigated the application of bacterial larvicides, Bacillus thuringiensis var. israelensis ( Bti ) and/or Bacillus sphaericus ( Bs ) for malaria vector control in SSA. Data for the review were identified through PubMed, the extensive files of the authors and reference lists of relevant articles retrieved. A total of 56 relevant studies were identified and included in the review. The findings indicated that, at low application rates, bacterial larvicide products based on Bti and/or Bs were effective in controlling malaria vectors. The larvicide interventions were found to be feasible, accepted by the general community, safe to the non-target organisms and the costs compared fairly well with those of other vector control measures practiced in SSA. Our review suggests that larviciding should gain more ground as a tool for integrated malaria vector control due to the decline in malaria which creates more appropriate conditions for the intervention and to the recognition of limitations of insecticide-based vector control tools. The advancement of new technology for mapping landscapes and environments could moreover facilitate identification and targeting of the numerous larval habitats preferred by the African malaria vectors. To build sustainable anti-larval measures in SSA, there is a great need to build capacity in relevant specialties and develop organizational structures for governance and management of larval source management programmes.

Background Long-lasting insecticidal nets (LLINs) are the primary method of malaria prevention. However, the widespread resistance to pyrethroids among major malaria vector species represents a significant threat to the continued efficacy of pyrethroid LLIN. Piperonyl butoxide (PBO) is a synergist that inhibits the activity of metabolic enzymes of the cytochrome P450 family known to detoxify insecticides including pyrethroids. Synergist LLIN incorporating PBO and a pyrethroid may provide improved control compared to pyrethroid-only LLIN. Methods The efficacy of VEERALIN® LN (VKA polymers Pvt Ltd, India), an alpha-cypermethrin PBO synergist net was evaluated in experimental huts in M’bé, central Côte d’Ivoire against wild pyrethroid resistant Anopheles gambiae  s.s. Comparison was made with a standard alpha-cypermethrin-treated net (MAGNet® LN, VKA polymers Pvt Ltd, India). Nets were tested unwashed and after 20 standardized washes. Results VEERALIN® LN demonstrated improved efficacy compared to MAGNet® LN against wild free-flying pyrethroid-resistant An. gambiae  s.s. Before washing, VEERALIN® LN produced mortality of An. gambiae  s.s. (51%) significantly higher than the standard pyrethroid-only net (29%) ( P  < 0.0001). Although there was a significant reduction in mortality with both LLINs after 20 washes, VEERALIN® LN remained superior in efficacy to MAGNet® LN (38 vs 17%) ( P  < 0.0001). Blood-feeding was significantly inhibited with both types of insecticide-treated nets relative to the untreated control net ( P  < 0.0001). Unwashed VEERALIN® LN induced significantly higher blood-feeding inhibition of An. gambiae  s.s. (62.6%) compared to MAGNet® LN (35.4%) ( P  < 0.001). The difference persisted after washing, as there was no indication that either LLIN lost protection against biting or blood-feeding. The level of personal protection derived from the use of VEERALIN® LN was high (87%) compared to MAGNet® LN (66–69%) whether unwashed or washed. The AI content of VEERALIN® LN after 20 washes decreased from 6.75 to 6.03 g/kg for alpha-cypermethrin and from 2.95 to 2.64 g/kg for PBO, corresponding to an overall retention of 89% for each compound. Conclusions The addition of the synergist PBO to pyrethroid net greatly improved protection and control of pyrethroid-resistant An. gambiae  s.s. The pyrethroid-PBO VEERALIN® LN has the potential to reduce transmission in areas compromised by pyrethroid resistance.

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases.

Increasing insecticide resistance in malaria vectors threatens the efficacy of current control tools, however knowledge of metabolic and cuticular mechanisms is widely lacking in Ghana. We examined the metabolic and cuticular resistance mechanisms in Anopheles gambiae mosquitoes from coastal and sahel zones of Ghana. WHO susceptibility tests and synergist assays were performed on F0 field collected An. gambiae s.l. Gene expression profiles of eight key metabolic and cuticular genes were determined using qRT-PCR. Moderate to high pyrethroid resistance (< 70%) were observed across all the sites. Piperonyl butoxide significantly increased susceptibility to pyrethroids across all sites and insecticides, implicating P450s. Gene expression analysis revealed overexpression of metabolic and cuticular resistance genes in field An. gambiae populations compared to the susceptible Kisumu strain. CYP6M2 and CYP6P3 were the most overexpressed metabolic genes in pyrethroid-resistant mosquitoes, compared to the pyrethroid susceptible mosquitoes in the coastal (FC: 122.28 and 231.86, p  < 0.05) and sahel (FC: 344.955 and 716.37, p  < 0.001) zones respectively. CYP4G16 (previously associated with cuticular resistance) was significantly overexpressed in only resistant mosquitoes (FC: 3.32–30.12, p  < 0.05). Overexpression of metabolic and cuticular resistance genes in local malaria vectors highlights the need to intensify insecticide resistance management strategies to control malaria in Ghana.

Background The World Health Organization (WHO) recommends mass distribution of insecticide-treated nets (ITNs) to prevent malaria transmission. Unfortunately, resistance to pyrethroids affects the efficacy of standard ITNs. To overcome this resistance and continue to protect the population, the WHO has recommended new types of ITNs that combine a pyrethroid insecticide with either a synergist (PBO) or a second insecticide, such as chlorfenapyr. This study examines the baseline characteristics of malaria vectors prior to the distribution of three types of insecticide-treated nets as part of a three-arm randomised controlled trial: Interceptor G2 (pyrethroid–chlorfenapyr), VEERALIN (pyrethroid–PBO), and MAGNet (pyrethroid only). Methods The study was carried out in 40 villages (grouped into 33 clusters) of Tiébissou district in central Côte d’Ivoire. To assess biting rate and biting behaviour, human landing catches were conducted hourly indoors and outdoors in six randomly selected houses in each cluster, starting at 18:00 and continuing until 08:00 the next morning. Adult mosquitoes collected were morphologically identified, and a subset of Anopheles gambiae sensu lato (s.l.) and An. funestus s.l. were speciated by quantitative PCR (qPCR). Plasmodium sporozoite infections were detected by qPCR to estimate infection rates. The entomological inoculation rate was calculated as the product of the mosquito biting rate and the sporozoite infection rate. Results Among the 10,698 mosquitoes collected, An. gambiae s.l. was the predominant species, accounting for 62.5% ( n  = 6683) of the catch, followed by An. funestus s.s., which accounted for 19.8% ( n  = 2120). Of the sub-sample of An. gambiae s.l. processed by PCR, 79.0% ( n  = 1291/1635) were An. coluzzii and the remaining were Anopheles gambiae s.s. Malaria vectors were highly aggressive, with an average of 14.8 bites/person/night for An. coluzzii , 2.0 b/p/n for An. gambiae s.s. and 5.4 b/p/n for An. funestus , representing an overall average of 22.2 b/p/n (95% CI 17.2–27.2 b/p/n). No significant difference was found in biting activity between indoor and outdoor environments ( Z  = −0.25, P  = 0.803). Plasmodium sporozoite infection rate was 2.4% (95% CI 1.3–3.6%) for An. coluzzii , 1.5% (95% CI 0.3–2.6%) for An. gambiae s.s. and 2.7% (95% CI 1.2–4.3%) for An. funestus . The estimated overall entomological inoculation rate was 0.4 infected b/p/n (95% CI 0.3–0.6) and varied between 0.0 and 0.2 infective bites/person/night according to species. There was no difference observed in entomological infection rate (EIR) between capture locations (indoors versus outdoors; Z  = 1.521, P  = 0.128). Conclusions This study shows that An. coluzzii and An. funestus were the main malaria vectors and showed similar biting patterns both indoors and outdoors. Anopheles funestus was found in high density in a limited number of villages. Malaria transmission was high despite universal distribution of pyrethroid-ITN in the district. Graphical Abstract

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with < 92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe/Mikalayi, but not between the latter two sample sites despite separation of approximately 800 km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population ( F ST ) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel ( Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation ( F ST ) meriting further exploration as a potential resistance associated marker.

Insecticide resistance in Anopheles gambiae s.l. threatens malaria vector control strategies in sub-Saharan Africa. Organophosphates such as pirimiphos-methyl have been deployed in Benin through Indoor Residual Spraying (IRS) since 2013 as alternatives to pyrethroids. However, no published evidence had documented resistance to this compound. This study provides the first confirmation of reduced susceptibility to pirimiphos-methyl in Benin and investigates potential resistance mechanisms. The study was conducted from January 2022 to December 2024. Larvae of An. gambiae s.l. were collected from 20 districts along a north–south transect of Benin and reared to adults under insectary conditions. Susceptibility tests were performed according to WHO protocols using 0.25% pirimiphos-methyl-impregnated papers, and 24-h mortality was recorded. Molecular assays were conducted for species identification and detection of the Ace-1R (G119S) mutation. A total of 1,744 females were tested. Full susceptibility was observed in eight districts. Suspected and confirmed resistance was detected in eight and four districts, respectively, including key IRS-treated areas. Molecular analysis identified An. coluzzii (47.7%), An. gambiae s.s. (47.9%), and An. arabiensis (4.4%). The Ace-1R mutation occurred at low frequencies (< 5%), suggesting a limited role of target-site resistance. This study provides the first evidence of pirimiphos-methyl resistance in An. gambiae s.l. populations in Benin. The findings underline the need for: (i) strengthened and geographically expanded resistance surveillance, (ii) rotation or replacement of pirimiphos-methyl with new chemistries such as clothianidin or chlorfenapyr, and (iii) reinforcement of integrated resistance management strategies to preserve the effectiveness of IRS and other vector control interventions. These recommendations are essential for supporting national malaria control programs in evidence-based decision-making.

Background Insecticide resistance poses a growing challenge to malaria vector control in Kenya and around the world. Following evidence of associations between the mosquito microbiota and insecticide resistance, the microbiota of Anopheles gambiae sensu stricto ( s.s .) from Tulukuyi village, Bungoma, Kenya, with differing permethrin resistance profiles were comparatively characterized. Methods Using the CDC bottle bioassay, 133 2–3 day-old, virgin, non-blood fed female F 1 progeny of field-caught An. gambiae s.s . were exposed to five times (107.5 µg/ml) the discriminating dose of permethrin. Post bioassay, 50 resistant and 50 susceptible mosquitoes were subsequently screened for kdr East and West mutations, and individually processed for microbial analysis using high throughput sequencing targeting the universal bacterial and archaeal 16S rRNA gene. Results 47 % of the samples tested (n = 133) were resistant, and of the 100 selected for further processing, 99 % were positive for kdr East and 1 % for kdr West. Overall, 84 bacterial taxa were detected across all mosquito samples, with 36 of these shared between resistant and susceptible mosquitoes. A total of 20 bacterial taxa were unique to the resistant mosquitoes and 28 were unique to the susceptible mosquitoes. There were significant differences in bacterial composition between resistant and susceptible individuals (PERMANOVA, pseudo-F = 2.33, P = 0.001), with presence of Sphingobacterium, Lysinibacillus and Streptococcus (all known pyrethroid-degrading taxa), and the radiotolerant Rubrobacter , being significantly associated with resistant mosquitoes. On the other hand, the presence of Myxococcus , was significantly associated with susceptible mosquitoes. Conclusions This is the first report of distinct microbiota in An. gambiae s.s . associated with intense pyrethroid resistance. The findings highlight differentially abundant bacterial taxa between resistant and susceptible mosquitoes, and further suggest a microbe-mediated mechanism of insecticide resistance in mosquitoes. These results also indicate fixation of the kdr East mutation in this mosquito population, precluding further analysis of its associations with the mosquito microbiota, but presenting the hypothesis that any microbe-mediated mechanism of insecticide resistance would be likely of a metabolic nature. Overall, this study lays initial groundwork for understanding microbe-mediated mechanisms of insecticide resistance in African mosquito vectors of malaria, and potentially identifying novel microbial markers of insecticide resistance that could supplement existing vector surveillance tools.

The age structure of a mosquito population helps estimate the proportion of vectors capable of transmitting malaria. Many malaria transmission models rely on mosquito longevity as key parameter. However, these are rarely measured in the field due to lack of a reliable and scalable age-grading method. An accurate method could improve predictions of malaria risk and the impact assessment of interventions. This study aimed to investigate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for malaria vector age-grading using insectary-reared and wild-caught mosquitoes. Anopheles gambiae s.s. mosquitoes were reared in the insectary to different known physiological and chronological ages to evaluate if MALDI-TOF MS could be used to distinguish between different age groups. Wild mosquitoes were collected from Mozambique and Kenya and dissected to determine their parity status. Reference spectra were obtained from mosquito’s cephalothorax and used to create predictive databases which were validated using independent samples. MALDI-TOF MS identified the physiological and chronological age of insectary-reared mosquitoes with 94.52% and 77% accuracy respectively. Field-collected mosquitoes were primarily An. funestus s.s. and An. gambiae s.s. Parity prediction accuracy was between 81% and 87%. MALDI-TOF MS was able to distinguish and differentiate mosquitoes based on their age structure (chronological and physiological) and parity status.

Background Entomopathogenic fungi like Metarhizium are emerging as effective biopesticides against malaria vectors. They reduce mosquito survival, fecundity, and flight ability, and reverse insecticide susceptibility in resistant Anopheles gambiae sensu lato strains. To elucidate the unclear underlying mechanisms, this study investigates the effects of fungal infections and insecticide exposure on the mosquito’s energy reserves and the expression of key metabolic and immune genes. Methods Three mosquito types: (i) pyrethroid-resistant An. gambiae sensu lato and two laboratory colonies: (ii) pyrethroid-resistant An. coluzzii VKPER and (iii) insecticide-susceptible An. gambiae sensu stricto Kisumu were used. They were infected with Metarhizium pingshaense S10 strain at a concentration of 10⁷ spores/mL (treatment groups) and with solvent only (0.05% Tween ® 80; control groups). Live mosquitoes were collected on days 0, 4, and 8 post-infection. They were used to quantify glucose, glycogen, and lipid via Van Handel’s protocol and to assess insecticide resistance. For resistance testing, mosquitoes underwent a standard WHO insecticide susceptibility test using deltamethrin (0.05%) or a control. Survival was measured 1 h after exposure, and surviving mosquitoes were analyzed by RT-qPCR for the expression of defensin and CYP6P3 , CYP6Z1 , and GSTe2 . Results Susceptible An. gambiae Kisumu were eliminated by deltamethrin, while resistant An. coluzzii VKPER and wild An. gambiae s.l. mosquitoes survived. However, deltamethrin exposure following Metarhizium infection significantly reduced survival in these resistant strains compared to the controls. This also resulted in reduced expression levels of defensin , GSTe2 , and CYP6Z1 compared to deltamethrin exposure alone, but no difference was found in the expression levels of CYP6P3 . These results collectively indicate that Metarhizium infection reduces mosquito survival by impairing their energetic reserves and ability to sustain vital physiological processes, including immune function and metabolic homeostasis. Conclusions We demonstrate that Metarhizium infection reverses insecticide resistance in An. gambiae s.l. by depleting energy reserves and suppressing the expression of detoxification genes. This mechanistic insight is crucial for optimizing the future integration of Metarhizium alongside conventional insecticides for malaria vector control. Graphical Abstract