Background Adult mosquito survival is a critical factor in malaria transmission because Anopheles vectors must live at least 11–14 days before they can transmit. However, there is no direct method to assess age. Traditional methods of mosquito age-grading rely on indirect proxies of reproductive history derived from dissection and observation of ovarian features (Detinova and Polovodova methods). Both approaches are labour-intensive, time-consuming, and demand specialized expertise; with little known about the extent to which are used in malaria surveillance programmes, and why they may be deprioritised. This study assessed current practices, capacity, and constraints related to mosquito age-grading for malaria vector surveillance in Africa, including knowledge and use of existing dissection-based methods and emerging alternatives such as infrared spectroscopy. Methods We conducted an online survey of 285 stakeholders from across Africa, including researchers, entomologists, and National Malaria Programme (NMP) personnel, to assess the practices, priorities, and barriers to mosquito age-grading. This was complemented with a series of in-depth interviews and focus group discussions to collect insights and perspectives from stakeholders regarding their familiarity and use of age-grading methods. Findings: More than 70% of survey respondents reported that malaria vector surveillance was routinely conducted by their institutions or countries, with the highest priority given to vector density, species identification, human biting rate, and insecticide resistance. Familiarity (awareness as opposed to knowledge) of age-grading methods was highest for the Detinova (55%) and Polovodova (44%), and lower for newer approaches, including infrared-spectroscopy. Only 50% of respondents indicated that they regularly assessed mosquito age (mostly Detinova method); with > 1/3 considering age-grading to be a high-priority in vector surveillance. Reported barriers to conducting mosquito-age-grading included insufficient technical expertise, perceived impracticality of ovary dissections for large-scale surveillance, inadequate tools, and limited funding. Conclusions Despite its critical role in malaria transmission, mosquito survival and age are rarely assessed in African vector surveillance programs. There is need for greater acknowledgment of these measures and their implications for disease transmission and control, and investment in tools, training, and funding to overcome current operational barriers. Integrating more practical and scalable age-assessment methods could enhance targeting of interventions.

Mating is a vital behavior for mosquito reproduction, yet it remains poorly understood under captive conditions. We examined the copulation dynamics of two key malaria vectors, Anopheles funestus, and Anopheles arabiensis, in controlled laboratory settings in Tanzania. We observed how variations in mosquito age and artificial lighting influence mating success for these two mosquito species within cages under controlled conditions. We conducted observations in 24-hour cycles, monitoring copulation events and insemination in females. We used generalized linear mixed models (GLMMs) for statistical analyses to assess how environmental conditions influence mating behavior. We found that Anopheles arabiensis exhibited rapid copulation, with 32.4% of individuals mating by Day 3 post-emergence, while An. funestus showed delayed activity, reaching a similar mating rate by Day 8. The introduction of artificial red light significantly accelerated copulation in An. funestus but did not affect An. arabiensis. Dissection confirmed successful sperm transfer and mating plug delivery in over 92% of copulating pairs for both species. Mating occurred primarily at night, with distinct peaks at 22:00 for An. arabiensis and 23:00 for An. funestus. In conclusion, our findings reveal species-specific differences in reproductive behavior, which could improve the colonization of An. funestus, a species historically challenging to rear in captivity. These insights may also inform the development of new vector control technologies, such as sterile insect techniques and genetic-based approaches, that exploit mosquito mating behaviors. ### Competing Interest Statement The authors have declared no competing interest.

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 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. 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.

[This corrects the article DOI: 10.1016/j.parepi.2022.e00264.].

Background It is often assumed that the population dynamics of the malaria vector Anopheles funestus , its role in malaria transmission and the way it responds to interventions are similar to the more elaborately characterized Anopheles gambiae . However, An. funestus has several unique ecological features that could generate distinct transmission dynamics and responsiveness to interventions. The objectives of this work were to develop a model which will: (1) reconstruct the population dynamics, survival, and fecundity of wild An. funestus populations in southern Tanzania, (2) quantify impacts of density dependence on the dynamics, and (3) assess seasonal fluctuations in An. funestus demography. Through quantifying the population dynamics of An. funestus , this model will enable analysis of how their stability and response to interventions may differ from that of An. gambiae sensu lato. Methods A Bayesian State Space Model (SSM) based on mosquito life history was fit to time series data on the abundance of female An. funestus sensu stricto collected over 2 years in southern Tanzania. Prior values of fitness and demography were incorporated from empirical data on larval development, adult survival and fecundity from laboratory-reared first generation progeny of wild caught An. funestus . The model was structured to allow larval and adult fitness traits to vary seasonally in response to environmental covariates (i.e. temperature and rainfall), and for density dependency in larvae. The effects of density dependence and seasonality were measured through counterfactual examination of model fit with or without these covariates. Results The model accurately reconstructed the seasonal population dynamics of An. funestus and generated biologically-plausible values of their survival larval, development and fecundity in the wild. This model suggests that An. funestus survival and fecundity annual pattern was highly variable across the year, but did not show consistent seasonal trends either rainfall or temperature . While the model fit was somewhat improved by inclusion of density dependence, this was a relatively minor effect and suggests that this process is not as important for An. funestus as it is for An. gambiae populations. Conclusion The model's ability to accurately reconstruct the dynamics and demography of An. funestus could potentially be useful in simulating the response of these populations to vector control techniques deployed separately or in combination. The observed and simulated dynamics also suggests that An. funestus could be playing a role in year-round malaria transmission, with any apparent seasonality attributed to other vector species.

Background: In south-eastern Tanzania where insecticide-treated nets have been widely used for >20 years, malaria transmission has greatly reduced but remains highly heterogenous over small distances. This study investigated the seasonal prevalence of Plasmodium sporozoite infections in the two main malaria vector species, Anopheles funestus and Anopheles arabiensis for 34 months, starting January 2018 to November 2020. Methods: Adult mosquitoes were collected using CDC-light traps and Pmkopack aspirators inside local houses in Igumbiro and Sululu villages, where earlier surveys had found very high densities of An. funestus. Collected females were sorted by taxa, and the samples examined using ELISA assays for detecting Plasmodium circumsporozoite protein in their salivary glands. Results: Of 7859 An. funestus tested, 4.6% (n = 365) were positive for Pf sporozoites in the salivary glands. On the contrary, only 0.4% (n = 9) of the 2382 An. arabiensis tested were positive. The sporozoite prevalence did not vary significantly between the villages or seasons. Similarly, the proportions of parous females of either species were not significantly different between the two villages (p > 0.05) but was slightly higher in An. funestus (0.50) than in An. arabiensis (0.42). Analysis of the 2020 data determined that An. funestus contributed 97.7% of all malaria transmitted in households in these two villages. Conclusions: In contexts where individual vector species mediate most of the pathogen transmission, it may be most appropriate to pursue a species-focused approach to better understand the ecology of the dominant vectors and target them with effective interventions to suppress transmission. Despite the ongoing efforts on tackling malaria in the two study villages, there is still persistently high Plasmodium infection prevalence in local populations of An. funestus, which now carry similar to 97% of all malaria infections and mediates intense year-round transmission. Further reduction in malaria burden in these or other similar settings requires effective targeting of An. funestus.

BACKGROUND:In rural south-eastern Tanzania, Anopheles funestus is a major malaria vector, and has been implicated in nearly 90% of all infective bites. Unfortunately, little is known about the natural ecological requirements and survival strategies of this mosquito species. METHODS:Potential mosquito aquatic habitats were systematically searched along 1000 m transects from the centres of six villages in south-eastern Tanzania. All water bodies were geo-referenced, characterized and examined for presence of Anopheles larvae using standard 350 mLs dippers or 10 L buckets. Larvae were collected for rearing, and the emergent adults identified to confirm habitats containing An. funestus. RESULTS:One hundred and eleven habitats were identified and assessed from the first five villages (all < 300 m altitude). Of these, 36 (32.4%) had An. funestus co-occurring with other mosquito species. Another 47 (42.3%) had other Anopheles species and/or culicines, but not An. funestus, and 28 (25.2%) had no mosquitoes. There were three main habitat types occupied by An. funestus, namely: (a) small spring-fed pools with well-defined perimeters (36.1%), (b) medium-sized natural ponds retaining water most of the year (16.7%), and (c) slow-moving waters along river tributaries (47.2%). The habitats generally had clear waters with emergent surface vegetation, depths > 0.5 m and distances < 100 m from human dwellings. They were permanent or semi-permanent, retaining water most of the year. Water temperatures ranged from 25.2 to 28.8 °C, pH from 6.5 to 6.7, turbidity from 26.6 to 54.8 NTU and total dissolved solids from 60.5 to 80.3 mg/L. In the sixth village (altitude > 400 m), very high densities of An. funestus were found along rivers with slow-moving clear waters and emergent vegetation. CONCLUSION:This study has documented the diversity and key characteristics of aquatic habitats of An. funestus across villages in south-eastern Tanzania, and will form an important basis for further studies to improve malaria control. The observations suggest that An. funestus habitats in the area can indeed be described as fixed, few and findable based on their unique characteristics. Future studies should investigate the potential of targeting these habitats with larviciding or larval source management to complement malaria control efforts in areas dominated by this vector species.

BackgroundOutdoor and early evening mosquito biting needs to be addressed if malaria elimination is to be achieved. While indoor-targeted interventions such as insecticide-treated nets and indoor residual spraying remain essential, complementary approaches that tackle persisting outdoor transmission are urgently required to maximize the impact.Major malaria vectors principally bite human hosts around the feet and ankles. Consequently, this study investigated whether sandals treated with efficacious spatial repellents can protect against outdoor biting mosquitoes.MethodologySandals affixed with hessian bands measuring 48cm 2 treated with 6 ml, 10 ml and 12 ml of transfluthrin were tested in large cage semi-field and full field experiments. Sandals affixed with hessian bands measuring 240cm 2 and treated with 10 ml and 12ml of transfluthrin were also tested semi field experiments. Human landing catches (HLC) were used to assess reduction in biting exposure by comparing proportions of mosquitoes landing on volunteers wearing treated and untreated sandals. Sandals were tested against insectary reared Anopheles arabiensis mosquitoes in semi-field experiments and against wild mosquito species in rural Tanzania.ResultsIn semi-field tests, sandals fitted with hessian bands measuring 48cm 2 and treated with 12 ml, 10ml and 6ml transfluthrin reduced mosquito landings by 45.9%, (95% confidence interval (C.I.) = 28–59%), 61.1% (48%–71%), and 25.9% (9% - 40%) respectively compared to untreated sandals. Sandals fitted with hessian bands measuring 240cm 2 and treated with 12 ml and 10ml transfluthrin reduced mosquito landings by 59% (43 - 71%) and 64% (48 - 74%) respectively.In field experiments, sandals fitted with hessian bands measuring 48cm 2 and treated with 12 ml transfluthrin reduced mosquito landings by 70% (60% - 76%) against Anopheles gambiae s.l and 66.0% (59% - 71%) against all mosquito species combined.ConclusionTransfluthrin-treated sandals conferred significant protection against mosquito bites in semi-field and field settings. Further evaluation is recommended for this tool as a potential complimentary intervention against malaria. This intervention could be particularly useful for protecting against outdoor exposure to mosquito bites. Additional studies are necessary to optimize treatment techniques and substrates, establish safety profiles and determine epidemiological impact in different settings.

Effective malaria surveillance requires detailed assessments of mosquitoes biting indoors, where interventions such as insecticide-treated nets work best, and outdoors, where other interventions may be required. Such assessments often involve volunteers exposing their legs to attract mosquitoes [i.e., human landing catches (HLC)], a procedure with significant safety and ethical concerns. Here, an exposure-free, miniaturized, double-net trap (DN-Mini) is used to assess relationships between indoor–outdoor biting preferences of malaria vectors, Anopheles arabiensis and Anopheles funestus, and their physiological ages (approximated by parity and insemination states). The DN-Mini is made of UV-resistant netting on a wooden frame and PVC base. At 100 cm × 60 cm × 180 cm, it fits indoors and outdoors. It has a protective inner chamber where a volunteer sits and collects host-seeking mosquitoes entrapped in an outer chamber. Experiments were conducted in eight Tanzanian villages using DN-Mini to: (a) estimate nightly biting and hourly biting proportions of mosquitoes indoors and outdoors; (b) compare these proportions to previous estimates by HLC in same villages; and, (c) compare distribution of parous (proxy for potentially infectious) and inseminated mosquitoes indoors and outdoors. More than twice as many An. arabiensis were caught outdoors as indoors (p < 0.001), while An. funestus catches were marginally higher indoors than outdoors (p = 0.201). Anopheles arabiensis caught outdoors also had higher parity and insemination proportions than those indoors (p < 0.001), while An. funestus indoors had higher parity and insemination than those outdoors (p = 0.04). Observations of indoor-biting and outdoor-biting proportions, hourly biting patterns and overall species diversities as measured by DN-Mini, matched previous HLC estimates. Malaria vectors that are behaviourally adapted to bite humans outdoors also have their older, potentially infectious sub-populations concentrated outdoors, while those adapted to bite indoors have their older sub-populations concentrated indoors. Here, potentially infectious An. arabiensis more likely bite outdoors than indoors, while potentially infectious An. funestus more likely bite indoors. These observations validate previous evidence that even outdoor-biting mosquitoes regularly enter houses when young. They also demonstrate efficacy of DN-Mini for measuring indoor–outdoor biting behaviours of mosquitoes, their hourly biting patterns and epidemiologically relevant parameters, e.g., parity and insemination status, without exposure to volunteers. The trap is easy-to-use, easy-to-manufacture and affordable (prototypes cost ~ 100 US$/unit).

Anopheles funestus mosquitoes currently contribute more than 85% of ongoing malaria transmission events in south-eastern Tanzania, even though they occur in lower densities than other vectors, such as Anopheles arabiensis. Unfortunately, the species ecology is minimally understood, partly because of difficulties in laboratory colonization. This study describes the first observations of An. funestus swarms in Tanzania, possibly heralding new opportunities for control.