Since the turn of the century, long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have played a major role in malaria control. However, the effectiveness of these tools is declining due to the development of insecticide resistance and other factors, creating an urgent need for complementary strategies. Larval source management (LSM), through the application of biological larvicides or the autodissemination of larvicides by ovipositing female mosquitoes, offers alternative avenues to target malaria vectors. Predicting the effectiveness of such approaches requires a detailed understanding of oviposition site selection behaviour in female Anopheles gambiae sensu lato. This study investigated the oviposition strategy of female Anopheles arabiensis in relation to aquatic habitat size under semi-field conditions in south-central Tanzania. An array of twelve alternating small (30 cm diameter, 20 L capacity) and large (60 cm diameter, 40 L capacity) artificial larval habitats was established in two compartments of a semi-field system. In quadruplicated experiments, forty wild gravid An. arabiensis females were released into each compartment and allowed to oviposit in their preferred habitats. The resulting third-instar larvae were collected, counted and preserved in ethanol. Larvae from one replicate were subjected to DNA extraction and microsatellite genotyping. Sibship analysis using Bayesian-likelihood methods (COLONY software) was conducted to reconstruct individual female oviposition behaviour. Rather than distributing eggs in proportion to habitat size, An. arabiensis females significantly preferred smaller habitats. Sibship analysis showed that 49.2

Background: Anopheles funestus, a major vector of malaria in Africa, has proven difficult to colonize in laboratory settings, impeding research on its biology and control. After several attempts, our team recently succeeded in colonizing a strain of An. funestus from Tanzania (FUTAZ). The objective of this study was to analyse the key fitness and genotypic characteristics of these mosquitoes during multiple filial generations of laboratory adaptation and compare them to wild An. funestus from Tanzania and a pre-existing colony of An. funestus from Mozambique (FUMOZ). Methods: Measures of mating success (percentage of female mosquitoes inseminated), body size (wing length), fecundity (number of eggs laid per female), and insecticide susceptibility (percentage of 24-hour mortality after exposure to insecticides) were compared between the newly established colonies of Tanzanian An. funestus (FUTAZ colonies), the long-established FUMOZ colonies, and a colony of Anopheles arabiensis maintained in the same laboratory. The maternal lineages of the An. funestus mosquitoes were investigated through a hydrolysis probe analysis of their mitochondrial DNA to identify distinct clades, I and II. Additionally, other intragenomic variations were examined through a PCR analysis of restriction fragment length polymorphisms (RFLP) on the third domain of 28S ribosomal DNA. These molecular markers were used to compare the FUTAZ colonies, FUMOZ colonies in Tanzania and South Africa, and the wild-collected An. funestus from Tanzania. Result: The mating success and body size of FUTAZ females declined significantly from filial generations F1 to F6 relative to the founder population (F0), but then increased from F7 onwards eventually matching FUMOZ by F9. Fecundity was similar across all colonies tested. However, it took significantly longer for 50% of the females in the FUTAZ and FUMOZ colonies (over 10 days) to mate compared to females in the An. arabiensis colony (approximately 5 days). Insecticide resistance appeared to be lost during colonization, but this varied with insecticide classes. Majority of mosquitoes in the FUTAZ colony, as well as the wild-caught Tanzanian An. funestus belonged to Clade I (80.4-89.4%) and RFLP type "Y" (90.5-91.4%), while the FUMOZ colonies were mostly Clade II (65.5-88.5%) and RFLP type "MW" (90.5-91.5%). Conclusion: This study suggests that the mating success and body size of An. funestus decreases significantly during the early stages of colonization, then increase as the mosquitoes adapt to laboratory conditions. It is therefore crucial to have a large enough founder population to persist through these early generations in order to achieve stable colonization of An. funestus. The Clade and RFLP genotyping demonstrated the genetic similarities between the FUTAZ mosquitoes and wild-caught Tanzanian An. funestus, but also showed that the new colony can be distinguished from the FUMOZ colony. ### Competing Interest Statement The authors have declared no competing interest.

Despite Anopheles funestus s.s. being a highly competent and widespread malaria vector in Africa, its population structure remains largely understudied in many countries, including Tanzania. Herein, we examine the genetic diversity, geographic isolation, and gene flow of An. funestus populations across ten administrative regions in mainland Tanzania. We employed 12 previously used microsatellite DNA markers to describe genetic diversity, isolation by distance, and gene flow patterns among ten An. funestus s.s. populations (n = 654) and one An. parensis population (n = 28), used as an outgroup, sampled across ten regions in mainland Tanzania. Overall, allelic richness (Na) and genetic diversity (HS) did not differ significantly among populations. Although some loci and populations showed significant departures from Hardy–Weinberg equilibrium, the patterns were not indicative of substructuring within locations. Pairwise genetic divergence (FST) values indicated clear separation between An. parensis and An. funestus s.s., with values exceeding 0.2, consistent with species-level differentiation. Among An. funestus s.s. populations, the highest divergence was observed between southeastern coastal populations (Mtwara, Ruvuma, Lindi) and inland populations, with FST values up to 0.288. There was no evidence of isolation by distance. Instead, patterns of genetic divergence suggested connectivity across the Rift Valley and heterogeneity among southeastern populations. Neighbor-joining analysis and Bayesian genotype clustering identified three distinct population groups: (i) An. parensis (Dodoma), (ii) a genetically distinct An. funestus s.s. population from Mtwara, and (iii) a more homogeneous cluster comprising the remaining An. funestus s.s. populations. Notably, the Mtwara population appeared highly differentiated, with divergence approaching that between An. funestus s.s. and An. parensis, supporting its distinctiveness but not undermining the role of An. parensis as an outgroup. Except for the Mtwara population, whose status will need to be clarified through whole-genome sequencing, moderate genetic divergence was found among An. funestus s.s. populations across Tanzania, despite geographical separation and the Rift Valley. The observed genetic structure suggests that anthropogenic gene flow may play a key role in shaping population divergence. Future studies should aim to delineate the effects of local adaptation from recent gene flow to further explore these dynamics

Anopheles funestus, a major vector of malaria in Africa, has proven difficult to colonize in laboratory settings, impeding research on its biology and control. After several attempts, our team recently succeeded in colonizing a strain of An. funestus from Tanzania (FUTAZ). The objective of this study was to analyze the key fitness and genotypic characteristics of these mosquitoes during multiple filial generations of laboratory adaptation and compare them to wild An. funestus from Tanzania and a pre-existing colony of An. funestus from Mozambique (FUMOZ). Measures of mating success, body size, fecundity, and insecticide resistance were compared between the newly established FUTAZ colonies, the long-established FUMOZ colonies, and a colony of Anopheles arabiensis maintained in the same laboratory. The maternal lineages of the An. funestus mosquitoes were investigated through a hydrolysis probe analysis of their mitochondrial DNA to identify distinct clades. Other intragenomic variations were examined by D3-RFLP genotyping of 28S ribosomal DNA. These molecular markers were used to compare the FUTAZ colonies, FUMOZ colonies in Tanzania (TZ) and South African (SA) laboratories, and the wild-collected Tanzanian An. funestus. The mating success and body size of FUTAZ females declined significantly from filial generations F1 to F6 relative to the founder population (F0) then increased from F7 onwards, eventually matching FUMOZ by F9. Fecundity was similar across all colonies tested. However, it took significantly longer for 50

Malaria remains a major global health challenge, with over 263 million cases and nearly 600,000 deaths reported in 2023, the majority in sub-Saharan Africa. While conventional interventions such as insecticide-treated nets, indoor residual spraying and antimalarial drugs have reduced transmission, progress has stalled due to the limitations of these interventions and the emergence of resistance. Gene drive-modified mosquitoes represent a promising, potentially transformative vector control strategy, capable of spreading malaria-refractory traits or suppressing mosquito populations. Successful field deployment will depend upon monitoring systems to track the presence and frequency of gene drive constructs as they spread and persist. Current molecular surveillance techniques, though effective, are resource-intensive and reliant on laboratory infrastructure and technical competencies. Here, we make the case for a near-universal and low-cost rapid diagnostic test (RDT) designed to detect gene drive mosquitoes in the field, to complement existing surveillance infrastructure. Two use cases are outlined: i) to detect the presence of the drive construct in a new population, and ii) to provide an estimate of drive frequency prior to more accurate laboratory-based measurements. We provide a target product profile for the RDT outlining minimally essential and ideal characteristics, including test procedures, sensitivity, specificity, usability by a range of stakeholders in field settings, and compatibility with pooled testing of mosquito samples. An RDT for gene drive construct detection would support community access and participation in monitoring, enhance regulatory oversight, and promote transparency in field trials, thereby facilitating responsible deployment of gene drive-based malaria interventions.

Members of the Anopheles gambiae complex, such as Anopheles gambiae sensu stricto (An. gambiae s.s.), Anopheles coluzzii and Anopheles arabiensis, are among the key malaria vectors in sub-Saharan Africa. These species are often abundant in areas of intense rice and maize farming with temporary water pools reflecting the dependence of their larvae on the pollen shed in such pools as food. In this study we explored the oviposition preference of wild-caught gravid An. arabiensis in response to maize and rice pollen in artificial aquatic habitats in a semi-field system. Twelve experimental breeding habitats were established in each of the two large compartments of a semi-field system. Rice or maize pollen was added into eight randomly selected habitats in eachcompartment; the remaining four habitats of each compartment were used as control habitats without pollens. In the first experiment, 40 gravid An. arabiensis were released in each compartment and left overnight to choose egg-laying habitats, following which the eggs were sampled and counted. The second experiment differed from the first experiment only in that the counted eggs were returned to the respective habitat where the development of the resultant larvae was monitored and recorded until pupation. Pollen types strongly affected the oviposition behaviour of gravid An. arabiensis. Females preferred to lay eggs in habitats with rice pollen on the water surface over those with maize pollen, and in habitats with maize pollen on the water surface over pollen-less controls. The development of larvae was significantly affected by the type of pollen in the habitats. The highest total number of An. arabiensis offspring were produced in habitats with rice pollen compared to those with maize pollen and no pollen. However, larval development success was comparatively lower in habitats containing rice pollen than those with maize pollen and no pollen, suggesting that the habitats with rice pollen were overcrowded. This study demonstrates that pollen types on the surface of aquatic habitats influence the oviposition site selection behaviour of gravid An. arabiensis and has carry-over effect on the developmental success of their offspring.

Abstract Background Sugar feeding is an essential aspect of mosquito biology that may be exploited for mosquito control by adding insecticides to sugar attractants, so-called ‘attractive targeted sugar baits’ (ATSBs). To optimize their effectiveness, ATSB products need to be maximally attractive at both short and long range and induce high levels of feeding. This study aimed to assess the attractiveness and feeding success of Anopheles mosquitoes exposed to attractive sugar baits (ASBs). Method Experiments were conducted in 2 × 5 × 2-m cages constructed within the semi-field systems (SFS) at Ifakara Health Institute, Bagamoyo, Tanzania. Male and female Anopheles gambiae s.s. and An. funestus s.s. mosquitoes were exposed to either 20% sucrose or different ASB station prototypes produced by Westham Co. in either (1) no-choice experiments or (2) choice experiments. Mosquitoes were exposed overnight and assessed for intrinsic or relative olfactory attraction using fluorescent powder markers dusted over the ASB stations and 20% sucrose and for feeding using uranine incorporated within the bait station and food dye in 20% sucrose controls. Results Both male and female An. gambiae and An. funestus mosquitoes were attracted to the ASBs, with no significant difference between the sexes for each of the experiments conducted. Older mosquitoes (3–5 days) were more attracted to the ASBs (OR = 8.3, [95% CI 6.6–10.5] P < 0.001) than younger mosquitoes (0–1 day). Similarly, older mosquitoes responded more to 20% sucrose (OR = 4.6, [3.7–5.8], P < 0.001) than newly emerged Anopheles. Of the four prototypes tested, the latest iteration, ASB prototype v1.2.1, showed the highest intrinsic attraction of both Anopheles species, attracting 91.2% [95% CI 87.9–94.5%]. Relative to ATSB v1.1.1, the latest prototype, v.1.2.1, had higher attraction (OR = 1.19 [95% CI 1.07–1.33], P < 0.001) and higher feeding success (OR = 1.71 [95% CI 1.33–2.18], P < 0.001). Conclusions Data from these experiments support using ASBs v1.2.1, deployed in large-scale epidemiological trials, as it is the most attractive and shows the highest feeding success of the Westham prototypes tested. The findings indicate that future bioassays to evaluate ATSBs should use mosquitoes of both sexes, aged 3–5 days, include multiple species in the same cage or chamber, and utilize both non-choice and choice tests with a standard comparator.

Population genetic analysis of mosquitoes is important for understanding the distribution of insecticide resistance alleles, devising sustainable control approaches, and understanding how vector populations are structured in space. Anopheles funestus is the dominant malaria vector in most parts of East and Southern Africa. To better understand its population genetic structure in Tanzania, we sequenced the genomes of 334 individual An. funestus mosquitoes from 11 regions across the country. Signs of reduced migration between western and eastern cohorts across the semi-arid central region containing the Rift Valley suggest a partial barrier to gene flow between these populations. This was evidenced by population structure between the eastern and western cohorts, as well as asynchronous selective sweeps and copy number variant profiles at the Cyp9k1 gene and Cyp6p gene cluster. Eastern cohorts, despite having less diversity and greater inbreeding, also share genetic histories characterized by low genome-wide Fst values with those in the west. This suggests that the barrier to gene flow is porous and likely represents continuous spatial structure rather than a complete barrier to migration. The observed population disconnectedness should be considered for insecticide deployment, resistance management, and the rollout of novel genetic-based vector control approaches. These findings provide the most detailed study of Tanzanian An. funestus population structure and resistance genetics to date. Future research should examine the epidemiological relevance of this partial discontinuity in gene flow and whether these populations have different malaria transmission abilities.

Population genetic analysis of mosquitoes is becoming increasingly important for understanding the distribution of insecticide resistance alleles, devising sustainable insecticide-based vector control approaches, and how malaria vector populations are structured in space. Anopheles funestus is the dominant malaria vector in Tanzania and most parts of East and Southern Africa. To better understand its population genomic structure in Tanzania, we sequenced the genomes of 334 individual An. funestus mosquitoes from 11 administrative regions. We found two genetically differentiated populations; one inland and at high altitude (found in Katavi, Kagera, Kigoma, and Mwanza) and a second coastal, at low altitude (found in Pwani, Morogoro, Tanga, Ruvuma, Mtwara, Dodoma, and Lindi), with differences in genetic diversity and inbreeding. We found asynchronous selective sweeps, associated with insecticide resistance phenotypes, at the Cyp9k1 gene, and Cyp6p gene cluster, with distinct copy number-variant profiles between the coastal and inland populations. These results suggest that inland and coastal An. funestus populations have divergent histories, with the arid, central region of Tanzania, which also contains the Rift Valley being a possible barrier to gene flow. Such population disconnectedness should be considered for insecticide deployment, resistance management, and the rollout of novel genetic-based vector control approaches. These findings provide the most detailed study of Tanzanian An. funestus population structure and resistance genetics to date. Future research should examine the epidemiological relevance of this discontinuity in gene flow and whether these populations have different malaria transmission abilities.

BACKGROUND:Immature anophelines inhabit aquatic environments with diverse physicochemical properties and microorganisms. In insectary settings, ammonia accumulation in larval rearing trays can lead to high larval mortality. Bacterial communities in these trays may influence ammonia levels through nitrification and denitrification. While symbiotic bacteria are known to be crucial for nutrition, digestion, reproduction, and immune responses in anophelines, the microbial communities specifically associated with Anopheles coluzzii larvae have not been characterised. METHODS AND RESULTS:Building on a study examining ammonia-capturing zeolite and water changes for rearing Anopheles coluzzii, this research characterised the bacterial communities using 16S rRNA gene sequencing to identify species linked to larval survival and phenotypic quality. Functional filters were applied to identify bacteria related to ammonia nitrification and their impact on larval development. qPCR was used to validate the sequencing data for the 10 most significant bacteria. Water changes significantly reduced bacterial diversity and abundance, improving adult mosquito development and quality. In contrast, untreated trays showed a higher abundance of potentially harmful bacteria, adversely affecting development. Applying zeolite increased nitrifying bacteria presence, benefiting mosquito growth while lowering toxic bacteria levels-trends confirmed by qPCR. CONCLUSIONS:This study offers insights into the bacterial communities in mosquito larval-rearing water, highlighting species that could enhance ammonia nitrification and overall rearing success.

Mosquito-borne pathogens continue to cause tremendous suffering, morbidity and mortality. For many of these diseases, vector control remains the most effective approach. The development and deployment of effective and efficient mosquito control products and strategies require a profound understanding of mosquito behaviour. To study complex mosquito behaviour, automated video tracking of mosquito flight paths has proven to be a comprehensive approach, and several video tracking approaches have emerged in recent years, making the choice for a suitable system challenging. Here, we conducted a literature review by searching PubMed and Google Scholar, and we identified 66 publications focusing on mosquito video tracking, which made use of eight different systems. We then compared and scored those video tracking systems by assessing their performance in the laboratory as well as their potential suitability for tracking mosquito behaviour in a field setting. While all eight systems have produced valuable information on mosquito behaviour, for tracking mosquitoes in the field, ‘Braid’, ‘EthoVision XT’ and ‘Trackit3D’ appear to be the most suitable systems as they need small disk capacity and are well adaptable to different settings. However, the optimal choice will ultimately depend on the specifications required to answer a given research question, the financial resources available and user preferences.

Abstract Background Members of the Anopheles gambiae complex are major malaria vectors in sub-Saharan Africa. Their larval stages inhabit a variety of aquatic habitats in which, under natural circumstances, they are preyed upon by different taxa of aquatic macroinvertebrate predators. Understanding the potential impact of predators on malaria vector larval population dynamics is important for enabling integrated local mosquito control programmes with a stronger emphasis on biocontrol approaches. This study experimentally evaluated the predation efficacy and foraging strategy of three common aquatic macroinvertebrate predators of An. gambiae, diving beetles (Coleoptera), backswimmers (Hemiptera), and dragonfly nymphs (Odonata) in a semi-field system in South-Eastern Tanzania. Methods An array of alternating small and large basins used as aquatic habitats was created in two compartments of a semi-field system and filled with well water. Field-collected adult diving beetles, backswimmers or dragonfly nymphs were randomly assigned to these habitats and Anopheles arabiensis larvae were added as prey in half of the habitats. The number of mosquito larvae consumed, predator mobility across habitats and mortality were recorded at 24, 48 and 72 h. Results The presence of An. gambiae larvae in habitats significantly increased the survival of backswimmer and dragonfly nymphs, which are not mobile. In contrast, diving beetles survived well under any initial condition by preferentially flying away from habitats without prey to nearby larger habitats with prey. The larval predation rates of predacious diving beetle, backswimmer and dragonfly nymphs were stable over time at a mean of 3.2, 7.0 and 9.6 larvae consumed each day. Conclusion This study demonstrates that aquatic macroinvertebrate predators display adaptive foraging behaviour in response to prey presence and aquatic habitat size. It also confirms the ability of these predators to significantly reduce An. gambiae larval densities in aquatic habitats, thus their potential for consideration as additional biocontrol tools for mosquito population reduction.

Maintaining fitness is an important consideration when mosquitoes are mass-reared for the deployment of genetic interventions that are designed to suppress populations because released mosquitoes need to compete with wild-type mosquitoes. Late-hatching mosquitoes are more suitable for transportation to remote field sites. Here, we investigated the fitness of late-hatching phenotypes in Anopheles coluzzii. Selected lines of the VK strain (from Burkina Faso) were created through bidirectional selection for early and late hatching, over 20 generations. These were compared with each other and the established Mopti reference strain from Mali, reared in the lab for >16 years. Significant differences in life-history traits were found between Mopti and VK strains but few differences were found between the selected VK lines. Considering that late-hatching VK lines showed no evidence of fitness costs, our results suggest that the late selected VK lines, which start hatching after 4 days, are an alternative option for egg shipment for mass mosquito releases over the well-established Mopti that hatches within 2 days and has lower adult survival.

Introduction:Larval source management, particularly larviciding, is mainly implemented in urban settings to control malaria and other mosquito-borne diseases. In Tanzania, the government has recently expanded larviciding to rural settings across the country, but implementation faces multiple challenges, notably inadequate resources and limited know-how by technical staff. This study evaluated the potential of training community members to identify, characterize and target larval habitats of Anopheles funestus mosquitoes, the dominant vector of malaria transmission in south-eastern Tanzania. Methods:A mixed-methods study was used. First, interviewer-administered questionnaires were employed to assess knowledge, awareness, and perceptions of community members towards larviciding (N = 300). Secondly community-based volunteers were trained to identify and characterize aquatic habitats of dominant malaria vector species, after which they treated the most productive habitats with a locally-manufactured formulation of the biolarvicide, Bacillus thuringiensis var. israelensis. Longitudinal surveys of mosquito adults and larvae were used to assess impacts of the community-led larviciding programme in two villages in rural south-eastern Tanzania. Results:At the beginning of the program, the majority of village residents were unaware of larviciding as a potential malaria prevention method, and about 20 % thought that larvicides could be harmful to the environment and other insects. The trained community volunteers identified and characterized 360 aquatic habitats, of which 45.6 % had Anopheles funestus, the dominant malaria vector in the area. The preferred larval habitats for An. funestus were deep and had either slow- or fast-moving waters. Application of biolarvicides reduced the abundance of adult An. funestus and Culex spp. species inside human houses in the same villages, by 46.3 % and 35.4 % respectively. Abundance of late-stage instar larvae of the same taxa was also reduced by 74 % and 42 %, respectively. Conclusion:This study demonstrates that training community members to identify, characterize, and target larval habitats of the dominant malaria vectors can be effective for larval source management in rural Tanzania. Community-led larviciding reduced the densities of adult and late-stage instar larvae of An. funestus and Culex spp. inside houses, suggesting that this approach may have potential for malaria control in rural settings. However, efforts are still needed to increase awareness of larviciding in the relevant communities.

Abstract Background The Anopheles funestus group includes at least 11 sibling species, with Anopheles funestus Giles being the most studied and significant malaria vector. Other species, like Anopheles parensis, are understudied despite their potential role in transmission. This article provides insights into the biology and insecticide susceptibility of An. parensis, with observations of its densities in northwestern Tanzania. Methods Mosquitoes were collected in three villages in Misungwi district, northwestern Tanzania, using CDC light traps and battery-powered aspirators indoors and human-baited double net traps outdoors. Female Anopheles adults were morphologically sorted and identified by PCR, and a subset was tested by ELISA for vertebrate blood meal sources and Plasmodium sporozoite infections. Insecticide susceptibility was assessed using the WHO protocol (2nd edition, 2018). Unfed females were dissected to assess parity, gonotrophic status and insemination status, while blood-fed females were monitored for oviposition to estimate egg counts. The prevalence of An. parensis was generally < 24% across all sites, except in Ngaya village, where it unexpectedly constituted 84% of PCR-amplified An. funestus sensu lato. This species was present in both indoor and outdoor collections, yet the females exclusively fed on non-human vertebrates, with no human blood meals detected. Parity rates were approximately 49% for resting and 46% for host-seeking females, with slightly higher percentages of both parous and inseminated females in the dry season compared to the wet season. Most parous females had oviposited once or twice, with those in the dry season ovipositing significantly more eggs. The average wing length of female An. parensis was 2.93 mm, and there was no significant impact of body size on parity, fecundity or insemination. The An. parensis mosquitoes were fully susceptible to pyrethroids, carbamates, organophosphates and organochlorides. Results The prevalence of An. parensis was generally < 24% across all sites, except in Ngaya village, where it unexpectedly constituted 84% of PCR-amplified An. funestus sensu lato. This species was present in both indoor and outdoor collections, yet the females exclusively fed on non-human vertebrates, with no human blood meals detected. Parity rates were approximately 49% for resting and 46% for host-seeking females, with slightly higher percentages of both parous and inseminated females in the dry season compared to the wet season. Most parous females had oviposited once or twice, with those in the dry season ovipositing significantly more eggs. The average wing length of female An. parensis was 2.93 mm, and there was no significant impact of body size on parity, fecundity or insemination. The An. parensis mosquitoes were fully susceptible to pyrethroids, carbamates, organophosphates and organochlorides.. Conclusion This study offers insights into the behaviours and insecticide susceptibility of An. parensis. Primarily feeding on non-human hosts, An. parensis is less significant in malaria transmission than more anthropophilic vectors. Unlike the pyrethroid-resistant An. funestus sensu stricto, An. parensis remains fully susceptible to public health insecticides despite the use of insecticidal bed nets. These findings provide a foundation for future research and may inform control strategies targeting residual malaria transmission involving An. parensis. Graphical Abstract

Abstract Background Indoor residual spraying (IRS) is one of the most effective malaria control tools. However, its application has become limited to specific contexts due to the increased costs of IRS products and implementation programmes. Selective spraying—selective spray targeted to particular areas/surfaces of dwellings—has been proposed to maintain the malaria control and resistance-management benefits of IRS while decreasing the costs of the intervention. Methods A literature search was conducted to find (1) studies that assessed the resting behaviour of Anopheles mosquitoes and (2) studies that evaluated the impact of selective spraying on entomological and malaria outcomes. Additional articles were identified through hand searches of all references cited in articles identified through the initial search. A cost model was developed from PMI VectorLink IRS country programmes, and comparative cost analysis reports to describe the overall cost benefits of selective IRS. Results In some studies, there appeared to be a clear resting preference for certain Anopheles species in terms of the height at which they rested. However, for other species, and particularly the major African malaria vectors, a clear resting pattern was not detected. Furthermore, resting behaviour was not measured in a standardized way. For the selective spray studies that were assessed, there was a wide range of spray configurations, which complicates the comparison of methods. Many of these spray techniques were effective and resulted in reported 25–68% cost savings and reduced use of insecticide. The reported cost savings in the literature do not always consider all of the IRS implementation costs. Using the IRS cost model, these savings ranged from 17 to 29% for programs that targeted Anopheles spp. and 18–41% for programmes that targeted Aedes aegypti. Conclusions Resting behaviour is generally measured in a simplistic way; noting the resting spot of mosquitoes in the morning. This is likely an oversimplification, and there is a need for better monitoring of resting mosquitoes. This may improve the target surface for selective spray techniques, which could reduce the cost of IRS while maintaining its effectiveness. Reporting of cost savings should be calculated considering the entire implementation costs, and a cost model was provided for future calculations.

Background Attractive targeted sugar baits (ATSBs) are promising new interventions that can complement existing vector control tools. However, reproducible and quantitative information on the level of attractiveness of ATSBs under field conditions is needed. Therefore, we customized camera traps for close-up imaging. We integrated them into a rugged ATSB monitoring station for day and nighttime recording of mosquitoes landing on the bait. Methods The camera traps were evaluated in a semifield system and then in the field in rural Tanzania. In semifield experiments, camera traps were set up in large cages (2 m × 5 m × 2 m) to record mosquitoes landing on an attractive sugar bait (ASB), a blank ASB, or 20% sucrose (w/v). Next, 198 mosquitoes (33 males and 33 females of Anopheles arabiensis, An. funestus and Aedes aegypti) were released into each large cage and allowed to seek a sugar meal for 72 h with a camera recording images of the mosquitoes present on the ASB at 1-min intervals. In the field, 16 camera traps were set in 16 households, 7 with ASB attractant, 7 with ASB blank, and 2 with 20% sucrose (w/v). Human landing catch (HLC) was performed on the same nights as the camera trap recordings. Results Under semifield conditions, significantly more mosquitoes visited the ASBs than the blank baits, with An. funestus visiting more frequently than An. arabiensis. There were no significant differences between female and male An. arabiensis visits, but female An. funestus visited more than their conspecific males did. The duration of visits did not vary between the ASB and blank controls or between the mosquito species. Moreover, mosquitoes visited the ASB or sucrose equally, with An. arabiensis visiting the baits more than An. funestus. Compared with male mosquitoes, female mosquitoes visited the baits more often. There was no significant difference in visit duration between the species. In the field study, a mean of 70 An. arabiensis were caught per person per night on HLC, while 1 individual was caught per night on ASBs. There were significantly more visits by mosquitoes to the ASB than to the ASB blanks or sucrose solution, with more An. arabiensis visiting the baits than An. funestus or Culex quinquefasciatus. Significantly more females than males visited the baits of all the species. Again, the duration of visits was similar among An. arabiensis, An. funestus and C. quinquefasciatus. Aedes aegypti very rarely visited ASBs in the semifield experiments, and none were observed on baits in the field. Conclusions Using camera traps to record still images of mosquitoes on ASBs offers reliable, reproducible and quantitative information on their attractiveness in various environmental conditions. Thus, camera traps serve as effective tools for evaluating and improving ATSB technology. Graphical abstract

Background Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania. Methods Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms. Results The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y). Conclusions Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.

Background Anopheles gambiae continues to be widespread and an important malaria vector species complex in Uganda. New approaches to malaria vector control are being explored including population suppression through swarm reductions and genetic modification involving gene drives. Designing and evaluating these new interventions require good understanding of the biology of the target vectors. Anopheles mosquito swarms have historically been hard to locate in Uganda and therefore have remained poorly characterized. In this study we sought to identify and characterize An. gambiae s.l mosquito swarms in three study sites of high An. gambiae s.l prevalence within Central Uganda. Methods Nine sampling visits were made to three villages over a 2-year period. Sampling targeted both wet and dry seasons and was done for 2 days per village during each trip, using sweep nets. All swarm data were analysed using the JMP 14 software (SAS Institute, Inc., Cary, NC, USA), parametrically or non-parametrically as appropriate. Results Most of the An. gambiae s.s. swarms sampled during this study were single-species swarms. However, some mixed An. gambiae s.s. and Culex spp. mosquito swarms were also observed. Swarms were larger in the wet season than in the dry season. Mean swarm height ranged from 2.16 m to 3.13 m off the ground and only varied between villages but not by season. Anopheles gambiae mosquitoes were present in all three villages, preferred to swarm over bare ground markers, and could be effectively sampled by field samplers. Conclusions This study demonstrated that An. gambiae s.l swarms could be effectively located and sampled in South Central Uganda and provided in-depth descriptions of hitherto poorly understood aspects of An. gambiae local swarm characteristics. Swarms were found close to inhabited households and were greater in size and number during the rainy season. Anopheles gambiae s.s swarms were significantly associated with bare ground markers and were sometimes at heights over 4 m above the ground, showing a necessity to develop tools suitable for swarm sampling at these heights. While mixed species swarms have been reported before elsewhere, this is the first documented instance of mixed genus swarms found in Uganda and should be studied further as it could have implications for swarm sampling explorations where multiple species of mosquitoes exist. Graphical Abstract

Macroinvertebrate predators such as backswimmers (Heteroptera: Notonectidae), dragonflies (Odonata: Aeshnidae), and predatory diving beetles (Coleoptera: Dytiscidae) naturally inhabit aquatic ecosystems. Some aquatic ecosystems inhabited by these macroinvertebrate predator taxa equally form malaria vector larval habitats. The presence of these predators in malaria vector larval habitats can negatively impact on development, adult body size, fecundity, and longevity of the malaria vectors, which form important determinants of their fitness and future vectorial capacity. These potential negative impacts caused by aquatic macroinvertebrate predators on malaria vectors warrant their consideration as biocontrol agents in an integrated program to combat malaria. However, the use of these macroinvertebrate predators in malaria biocontrol is currently constrained by technical bottlenecks linked to their generalist predatory tendencies and often long life cycles, demanding complex rearing systems. We reviewed the literature on the use of aquatic macroinvertebrate predators for biocontrol of malaria vectors from the An. gambiae s.l. complex. The available information from laboratory and semi-field studies has shown that aquatic macroinvertebrates have the potential to consume large numbers of mosquito larvae and could thus offer an additional approaches in integrated malaria vector management strategies. The growing number of semi-field structures available in East and West Africa provides an opportunity to conduct ecological experimental studies to reconsider the potential of using aquatic macroinvertebrate predators as a biocontrol tool. To achieve a more sustainable approach to controlling malaria vector populations, additional, non-chemical interventions could provide a more sustainable approach, in comparison with the failing chemical control tools, and should be urgently considered for integration with the current mosquito vector control campaigns.