From the field to the laboratory: establishment of Anopheles funestus s.s. colony in Burkina Faso

From the field to the laboratory: establishment of Anopheles funestus s.s. colony in Burkina Faso | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article From the field to the laboratory: establishment of Anopheles funestus s.s. colony in Burkina Faso Grégoire Sawadogo, Saberé O.G. Yemien, Ibrahim Diallo, Odette N. Zongo, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9291181/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Anopheles funestus s.s plays a major role in sustaining residual malaria transmission in many African settings. Despite its epidemiological importance, research on its biology and developing effective control strategies is greatly hindered by the persistent challenges associated with the establishing of stable colonies in laboratory. This study capitalises a successful process for establishing An. funestus strain in laboratory population in Burkina Faso (FUNBF). Methods Adult female mosquitoes were collected over a two-year period in Diébougou and Soumousso using mouth and battery-powered electric aspirators. These mosquitoes were morphologically identified as belonging to the An. funestus group. Individual females were allowed to oviposit separately in 200mL disposable cups. Female mosquitoes were then confirmed at the molecular level as An. funestus s.s. Progeny were reared under standard laboratory conditions. Fecundity and egg hatch rate were recorded for founder (F0) females. Results A total of 1,871 female mosquitoes were collected across two years to establish the colony. F0 mean fecundity was 85± 2.9 eggs per female (range: 35–124) with a mean hatching rate of 88.6% (range: 66.4–100%). FUNBF has been maintained successfully for 23 generations with sustained egg production. Conclusion This work represents a significant advance in the domestication of An. funestus , one of the main malaria vectors in sub-Saharan Africa. Malaria vector Mosquito rearing Sub-Saharan Africa Residual malaria egg production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Malaria remains a major global health concern, with an estimated 247 million cases worldwide and nearly half of the global population at risk [ 1 ]. In sub-Saharan Africa, four dominant mosquito species ( An. gambiae s.s., An. arabiensis, An. coluzzii, and An. funestus s.s ) drive malaria transmission [ 2 , 3 ]. While extensive research has focused on the An. gambiae complex, An. funestus remains comparatively understudied, largely because it is exceptionally difficult to rear and maintain under laboratory conditions [ 4 ]. The An. funestus species complex comprises at least 13 recognized members, including An. aruni , An. brucei , An. confusus , An. funestus sensu stricto (s.s.), An. funestus -like, An. fuscivenosus , An. leesoni , An. longipalpis type A, An. longipalpis type C, An. parensis , An. rivulorum , An. rivulorum-like , and An. vaneedeni [ 5 – 8 ]. The vectorial capacity of these species varies, and only An. funestus s.s. contribute to the spread of malaria parasite in certain regions [ 9 , 10 ]. An. funestus s.s control is mainly based on the use of insecticide through impregnated nets. Unfortunately, the effectiveness of this control strategy has been compromised for different reasons [ 11 ]. This species exhibits strong anthropophilic and endophilic behaviour, high levels of insecticide resistance, and longer adult longevity than other major African malaria vectors [ 12 – 14 ]. These characteristics make it a highly efficient vector and an important driver of persistent malaria transmission, particularly in areas where control programmes depend largely on insecticide-based interventions [ 15 ]. Nevertheless, control failure in An. funestus is unlikely to be explained by insecticide resistance alone. Behavioural plasticity, including exophilic, exophagic, and partially zoophilic tendencies reported in some populations, may allow this species to evade contact with long-lasting insecticidal nets and indoor residual spraying [ 16 ]. In addition, because An. funestus often reaches high densities later in the transmission season and persists into the dry season through its exploitation of more permanent, vegetated breeding sites, its peak abundance may not fully coincide with the timing of control efforts that are often designed around the rainy-season dynamics of An. gambiae [ 17 ]. These combined behavioural, ecological, and physiological features likely contribute to the limited effectiveness of standard interventions against An. funestus and to its continuing role in residual malaria transmission. The establishment of laboratory mosquito colonies has been pivotal in advancing understanding of their biology, including developmental processes, mechanisms of insecticide resistance, and pathogen transmission dynamics. Such colonies also provide standardised conditions for evaluating novel insecticides and innovative vector control strategies, including gene drive tools [ 18 ]. Mosquito colonisation remains challenging, particularly for eurygamic species such as Anopheles funestus , which show limited mating success in confined laboratory conditions, low oviposition rates, and high sensitivity to environmental fluctuations such as temperature and humidity[ 20 , 21 ]. Attempts to colonise An. funestus dates back to the 1950s, when an initial effort in Nigeria failed after only a few months [ 22 ]. To date, only a few laboratory colonies have been successfully maintained for extended generations, including: the FANG strain (Funestus ANGola), the FUMOZ strain (Funestus MOZambique) and more recently the FUTAZ from Tanzania [ 8 , 23 ]. Given the broad geographic distribution of Anopheles funestus sensu stricto (a member of the An. funestus species complex) and the diversity of ecological and operational environments in which it occurs, significant genetic structuring may exist among populations. This variability may be shaped by local ecological adaptation, insecticide selection pressure, and levels of gene flow, resulting in differences in key vectorial traits across regions. It is therefore important to distinguish variation among geographically distinct populations of An. funestus s.s. from differences attributable to other, morphologically similar species within the complex. As a result, reliance on a single laboratory strain may not adequately represent the biological and epidemiological diversity of the species. The establishment of stable laboratory colonies from multiple geographic origins is therefore essential to support robust investigations of An. funestus biology, insecticide resistance, parasite–vector interactions, and the assessment of novel vector control strategies, including genetic control methods. In this study, we describe the establishment of the first Anopheles funestus laboratory colony from West Africa, derived from field populations collected in Burkina Faso. This represents an important advance, given that An. funestus is a major malaria vector in West Africa and that no colony from this region has previously been available. We detail the collection methods and colony maintenance protocols implemented to support successful establishment. By documenting these procedures, we provide practical guidance for laboratories seeking to colonise An. funestus , thereby strengthening research capacity on this ecologically and epidemiologically important vector and supporting future studies, including those aimed at developing locally relevant control strategies. Material and Methods Mosquito field collection sites Following our countrywide entomological surveillance in 2022 across about 70 districts, a few sites were identified as conducive for An. funestus occurrence [ 24 ]. Therefore, Anopheles adult mosquitoes were collected from two localities in western Burkina Faso, including Diébougou (10°57’41” N, 3°15’00” W) and Soumousso (11°00’46” N, 4°02’45” W) (Fig. 1 ). Diébougou is located 136 km south-east of Bobo-Dioulasso, the second-largest city in Burkina Faso. In these sites, several Anopheles species coexist, including An. gambiae s.s, An. coluzzii and An. funestus [ 24 ]. Previous studies have reported high densities of An. funestus toward the end of the rainy season and during the cool dry period [ 25 , 26 ]. These conditions make the area particularly suitable for sampling and for establishing An. funestus laboratory colonies. Soumousso is a typical village in the Guinean savannah, located 55 km east of Bobo-Dioulasso. The village is home to the primary malaria vectors: An. coluzzii , An. gambiae s.s, An. funestus , and An. nili . Additionally, An. arabiensis is occasionally found at low frequency, accounting for approximately 5% of An. gambiae s.l. samples [ 25 ]. Adult Anopheles funestus females used for colony establishment were collected in Soumousso and Diébougou. Bobo-Dioulasso indicates the location of the insectary where mosquitoes were maintained, and colonisation experiments were conducted. Mosquito collection period and handling Adult female mosquitoes were collected at the end of the rainy season (October to December) in 2023 and 2025 between 5 a.m. and 7 a.m. Mosquitoes were sampled both indoors and outdoors from inhabited and uninhabited houses and chicken coops, as well as from resting sites such as clay water jars and tree holes (Fig. 2 ). To maximise capture efficiency while preserving specimen viability, both mouth aspirators and battery-powered electric aspirators were used. The latter was operated at reduced voltage to reduce suction force, thereby minimising injury or mortality during mosquito collection. Mosquitoes were collected in cages measuring 30cm x 30cm x 30 cm. Once collection was complete, the cages were covered with wet mops and transported in an air-conditioned vehicle to the laboratory. All collected mosquitoes were morphologically identified to genus and species level using the identification keys of Gillies and Coetzee [ 26 ] then transported to the insectary at the Institut de Recherche en Sciences de la Santé (IRSS) for individual oviposition. Adult mosquitoes were collected using mouth aspirators inside uninhabited and inhabited houses ( A and B ). In some cases, mosquitoes were also collected in peri-domestic water storage containers ( C ). Anopheles funestus strain establishment Insectary conditions Adult mosquitoes were maintained under standard insectary conditions at 27 ± 2°C, and 75–80% relative humidity, the larvae were kept at 29 ± 2°C with a 12:12 h light-dark cycle [ 27 ]. Individual oviposition Individual female mosquitoes were isolated for oviposition in 200 mL disposable cups (Fig. 3 ). Each cup was lined with a strip of filter paper (15 × 5 cm) to provide a suitable substrate for egg deposition. Approximately 50 mL of deionised water was added to each cup to stimulate oviposition. The cups were covered with a fine mosquito net secured with an elastic band, incorporating a small central slit to allow careful introduction of females without injury. Each cup was assigned a unique identification code to enable accurate tracking of individual females and their progeny. A cotton pad soaked in a 10% glucose solution was placed on top of the netting and replenished daily to maintain adult survival during the oviposition period. All cups were kept under insectary conditions and were inspected daily to assess oviposition success. Females that laid eggs were removed and preserved individually in 1.5 mL Eppendorf tubes containing 80% ethanol, with tube labels corresponding to the unique identifiers of their respective cups. Eggs were carefully transferred to larval trays containing deionised water and maintained under insectary conditions until hatching. Larvae of An. funestus s.s PCR positive were pooled and reared to adulthood with finely ground TetraMin® fish food. Females were placed individually in cups covered with a fine mosquito net secured with an elastic band. Each cup was lined on the inner wall with a strip of filter paper to provide a suitable substrate for egg deposition. Approximately 50 mL of dechlorinated water was added to each cup to stimulate oviposition. Cups were monitored daily for oviposition. Molecular identification of founders Genomic DNA was extracted from single mosquitoes using DNazol protocol and analysed by multiplex Polymerase Chain Reaction (PCR) assay [ 28 ] to distinguish sibling species within the An. funestus group. PCR reactions were carried out in a final volume of 20 µl, consisting of 4 µl of 5X FIREPol® Master Mix,11.9 µl of nuclease-free water, 0.3 µl of each of the five species-specific primers, and 2 µl of genomic DNA template. Thermal cycling conditions consisted of an: initial denaturation at 94°C for 10 min; 35 cycles of denaturation at 94°C for 45 s, annealing at 45°C for 30 s, and extension at 72°C for 40 s; followed by a final extension at 72°C for 10 min [ 28 ]. PCR products were resolved by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualised under UV illumination. Amplicon sizes were compared against a 100bp molecular size marker and existing positive controls of An. funestus s.s to determine species identity. Mosquitoes were collected in the field using aspirators (1), transported alive to the insectary (2) and morphologically identified under a stereomicroscope (2). Individual oviposition assays were conducted to obtain single-family egg batches (4). Species identity of parental females was confirmed by PCR (5), and only Anopheles funestus s.s lines were retained (6). Larvae from confirmed females were pooled and reared to adulthood (7), enabling colony establishment and maintenance across generations (8). Colony maintenance and challenges beyond the F1 generation Larval and adult feeding regimes Larvae from the F1 generation onwards were maintained under standard insectary conditions (see Insectary conditions section above) in rectangular trays (30 cm × 24 cm × 6 cm) containing 1 L of deionised water. Finely ground TetraMin® fish food was provided as the larval diet (Table 1 ). Adults were maintained on a 10% glucose solution provided ad libitum , with a cotton pad soaked daily. The first blood-feeding occurred four days after the last adult emergence. The interval between the first and second blood-feeding ranged from two to four days. During colony establishment, blood-feeding was attempted at 2:00 pm, 4:00 pm, 6:00 pm, 7:00 pm, and 9:00 pm using the Hemotek system with rabbit blood. As these preliminary observations were not part of a formal experiment, no comparative analysis was performed; 7:00 pm was selected for subsequent blood-feeding. Mosquitoes were starved for 5 hours before each blood feed. Table 1 Larvae feeding schedule for 250 larvae in a tray Larval age mg per larva mg per tray Feeding schedule Day 0 (Floating) 0.12 30 1 drop Day1 (splitting) 0.12 30 1 drop Day 2 0.12 30 1 drop Day 3 0.12 30 1 drop Day 4 0.18 45 1 drop + ½ drop Day 5 0.18 45 1 drop + ½ drop Day 6 0.18 45 1 drop + ½ drop Day 7 0.18 45 1 drop + ½ drop, Day 8 0.18 45 1 drop + ½ drop Day 9 0.24 60 2 drops Day 10 0.24 60 2 drops Day 11 0.3 75 1 drop + ½ drop 1 drop = 30mg Reproductive performance and oviposition challenges A major difficulty in colony maintenance was that An. funestus females did not readily lay eggs under standard insectary conditions. To stimulate oviposition, several strategies were tested to reproduce natural breeding cues. These included providing moistened cotton pads in Petri dishes, placing bowls filled with tap water, filtered larval water, and, in some cases, adding fresh grass stems to the bowls. During routine colony management two consecutive blood meals were required. Four days after the second blood meal, oviposition bowls were placed in the adult cages. Each bowl contained deionised water with filter paper partially immersed along the sides and supplemented with a small quantity of fresh grass. Eggs were then transferred to trays and larvae were maintained on ground TetraMin® with feeding quantities adjusted according to larval developmental stage and their quantity (Table 1 ). Mosquitoes were blood-fed at 4 and 6–8 days post-emergence (1). Eggs bowls were placed 48 hours post blood-feeding and eggs were collected after 72 hours (2), hatched within 2 days (3), and larvae developed into pupae in about 2 weeks (4). Adults emerged 48 hours later (5), marking the start of a new cycle. Fecundity and fertility assessment To assess An. funestus fecundity and fertility in laboratory condition, females were allowed to individually lay eggs in oviposition cups. Among the females that successfully laid eggs, 48 were randomly selected for fecundity and hatching assessment. The total number of eggs laid by each female was counted under a stereomicroscope. Oviposition cups were inspected daily, and upon hatching, first-instar larvae were counted and transferred to a new rearing container. The original oviposition cups were subsequently monitored for an additional three days to record any delayed hatching. Results Wild-caught mosquitoes for colony establishment . Adult mosquitoes were collected from October to December 2023 and 2025 in Soumousso and Diebougou to support the establishment of An. funestus laboratory strain in the insectary. Female mosquitoes were collected alive at each site, transferred to cages (30cm × 30cm × 30cm), fed with a 10% glucose solution and transported to the laboratory for colony establishment. A total of 1,871 mosquitoes were morphologically identified as An. funestus s.l . Of these, 794 females laid eggs, corresponding to a 42.44% success rate across collections (Table 2 ). Table 2 Summary of Anopheles funestus collections and oviposition outcomes by collection year Collection_years Number_collected Total_laid Total_unlaid %Laid %Unlaid 2023 760 438 322 57.64 42.36 2025 1,111 356 755 32.04 67.95 Total 1,871 794 1,077 42.44 57.56 Colony maintenance To ensure successful colony maintenance, several procedural adjustments were implemented. Blood feeding was standardized at 7:00 pm and performed twice prior to oviposition. In addition, oviposition success improved when females were provided with bowls lined with filter paper and supplemented with fresh grass, which appeared to stimulate egg laying. Fecundity and hatching success of founder An. funestus females Female reproductive performance showed variability across individuals. The number of eggs laid per female ranged broadly, with a mean fecundity of 85± 2.9 eggs per female. Despite this variability in egg production, fertility remained high, as hatching rates were consistently elevated across females, with most individuals showing hatching success above 80%. (A) number of eggs laid per female and (B) hatching rate per female. Each point represents an individual female; horizontal bars indicate the mean ± standard error. Discussion The establishment of laboratory colonies of malaria vectors is fundamental to research on their biology, behaviour, and control. Such colonies enable consistent, controlled evaluation of insecticides and novel control strategies, while also providing insight into variation in vector susceptibility and resistance mechanisms. In this context, successful colonisation represents an important step toward generating reproducible experimental material for studies that are difficult to conduct using field-caught mosquitoes alone. This study demonstrates the feasibility of establishing a local colony of An. funestus s.s. in Burkina Faso, a species recognised as difficult to maintain in captivity due to its sensitivity to rearing conditions, and low egg-laying rate in the laboratory [ 8 , 23 ]. The colony is successfully maintained up to generation 23, illustrating the effectiveness of the strategies implemented to optimise collection, egg laying, and rearing. Field collection methods were specifically adapted to maximize mosquito survival for colonization purposes. Mouth aspirators, although limited in throughput, allowed the collection of live females with minimal physical damage. In parallel, battery-powered mechanical aspirators were used at reduced suction power to increase collection efficiency while limiting injury-induced mortality. The combination of these approaches enabled the collection of more than 2,000 live specimens, with 1,871 individuals morphologically identified as belonging to An. funestus group. Transport from field sites to the insectary represents a critical bottleneck in colony establishment, particularly for An. funestus , a species known to be sensitive to environmental stress[ 29 , 30 ]. Mortality during transport is commonly associated with desiccation and heat stress, especially under tropical field conditions. To mitigate these effects, transport cages were covered with wet mops to maintain humidity, and vehicle air conditioning was used to reduce thermal stress. Such measures are consistent with established recommendations for maintaining field-collected Anopheles under optimal survival conditions prior to insectary processing [ 31 ]. These operational adaptations were essential to preserve female viability and ensure successful downstream oviposition, thereby contributing to the overall colonization effort. Molecular identification of ovipositing females revealed that the majority belonged to An funestus s.s in both sampling years. In 2023, 85.6% of egg-laying females were confirmed as An. funestus s.s ., while in 2024 this proportion decreased to 77.8%. These findings indicate that although An. funestus s.s . was the predominant species among reproducing females, a non-negligible proportion of morphologically identified specimens belonged to other members of the An. funestus group. Successful colonization of An. funestus has historically been constrained by its behavioral and ecological specificity, requiring careful optimisation of insectary conditions [ 29 , 30 ]. In this study, sustained colony maintenance was achieved after procedural adjustments targeting blood feeding and oviposition behavior. Standardizing blood feeding at 7:00 pm and providing two consecutive blood meals prior to oviposition likely aligned feeding with the species’ natural nocturnal biting rhythm, thereby enhancing blood feeding rates and subsequent egg development. The strong association between circadian feeding patterns and reproductive output has been well documented in Anopheles mosquitoes[ 31 ]. In addition, oviposition success improved when females were provided with bowls lined with filter paper and supplemented with fresh grass. An. funestus is known to preferentially oviposit in permanent or semi-permanent water bodies with emergent vegetation, unlike species such as An. gambiae that exploit temporary habitats [ 5 , 32 ]. The incorporation of grass likely mimicked natural breeding-site cues, providing tactile or chemical stimuli that triggered egg laying. Similar substrate-dependent oviposition behavior has been reported in laboratory and semi-field studies of the An. funestus group [ 30 ]. Egg-laying rates of 42.44% combined with the high proportion of engorged females, suggest that mosquitoes acquire blood meals primarily within human dwellings before resting in nearby uninhabited structures. This resting behaviour highlights potential limitations of vector control strategies relying exclusively on indoor residual spraying. Similar patterns have been reported previously in the same area, particularly in Soumousso, where approximately 40% of An. funestus were collected outdoors compared to 60% indoors [ 25 ], supporting the presence of flexible resting behaviour in this species. Beyond these operational parameters, we assessed fecundity and fertility, two key indicators not only for establishing laboratory strains but also for implementing vector control strategies such as gene drives and the sterile insect technique. Our results showed a mean fecundity of 85±2.9 eggs per female and a mean hatching rate of 88.62%. These values are broadly comparable to those reported for the FUMOZ strain (76 eggs per female) and the recently established FUTAZ strain from Tanzania (64 eggs per female) [ 8 ]. However, it is important to note that in the present study, these parameters were evaluated using mosquitoes collected directly from the field (F0 generation). Differences in fecundity and fertility may therefore reflect ecological, physiological, or nutritional factors associated with field-collected females, as well as the absence of laboratory induced constraints observed in long-term colonised strains. Consequently, these results should be interpreted as indicative of the reproductive potential of field-derived An. funestus rather than as direct measures of long-term colony performance. Taken together, these findings demonstrate that An. funestus populations from Burkina Faso exhibit high reproductive potential and behavioural flexibility, both of which are critical for successful early laboratory colonisation. The ability to obtain eggs beyond the F0 generation, combined with the high fecundity and hatching rates observed in field-derived females, highlights the feasibility of establishing a stable laboratory strain when key operational conditions are met. Although further optimisation and controlled evaluations across successive generations will be required, particularly to assess mating success and long-term colony performance, these results provide a practical framework for future colonisation efforts. Importantly, establishing a local An. funestus strain would support downstream applications, including insecticide resistance studies, behavioural assays, and the evaluation of novel vector control strategies. Although our study was broadly successful, there were some limitations. Albeit the colony was established initially, several parameters used during colony maintenance, such as adding grasses in oviposition bowls to stimulate egg-laying and adjusting blood-feeding schedules, were applied empirically and may require further optimisation. Previous work has shown that oviposition cues, blood-feeding timing, and microenvironmental structure can strongly influence the reproductive performance of An. funestus and other eurygamic species [ 33 ], suggesting that a more systematic evaluation of these factors will be essential to improve colony stability. Future studies should refine these conditions and explore alternative stimuli that better support consistent oviposition and successful progression to subsequent generations. Conclusion This study report successful colonisation of An. funestus in western Burkina Faso. Considering the inherent difficulty of establishing this species under laboratory conditions, our observations traced these factors back to field collection, revealing that An. funestus rest not only in inhabited houses but also abundantly in uninhabited houses. Colony maintenance required specific adjustments, notably blood feeding at 7:00 pm and the use of oviposition bowls containing fresh grasses. Further investigations are required to characterise this strain, particularly its insecticide resistance profile, and to optimise critical parameters such as larval nutrition, which remains essential for sustainable colony maintenance. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests All the authors have read and accepted this version of the manuscript. The authors also declare there are no conflicts of interest. Funding This work was supported by the Bill & Melinda Gates Foundation (INV037164). Author Contribution G.S, S.O.G.Y, O.N.Z, M.K, H.K, A.A, N.T, I.D, H.M and AD conceived the study. G.S, S.O.G.Y, O.N.Z, H.K, A.A and S.I performed the field collection. G.S, S.O.G.Y, O.N.Z, S.C, B.O, A.A, H.K and I.D performed experiments. G.S performed the data analyses. G.S wrote the early version of the manuscript. A.D secured the funding acquisition. S.O.G.Y, O.N.Z, M.K, H.K; N.T, H.M, H.H, A.L, T.N and A.D edited and validated the manuscript. Acknowledgement The authors would like to acknowledge with appreciation the valuable collaboration and technical support provided by the Institut de Recherche en Sciences de la Santé, Direction Régionale de l’Ouest (IRSS-DRO, Burkina Faso). Availability of data and materials Data are provided within the manuscript or supplementary information files References WHO. World malaria report 2023 [Internet]. 2023 [cited 2024 Aug 23]. Available from: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023 Coetzee M, Koekemoer LL. Molecular systematics and insecticide resistance in the major African malaria vector Anopheles funestus. Annu Rev Entomol. 2013;58:393–412. doi: 10.1146/annurev-ento-120811-153628 PubMed PMID: 23317045. Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, et al. A global map of dominant malaria vectors. Parasit Vectors. 2012;5:69. doi: 10.1186/1756-3305-5-69 PubMed PMID: 22475528; PubMed Central PMCID: PMC3349467. Hunt RH, Brooke BD, Pillay C, Koekemoer LL, Coetzee M. Laboratory selection for and characteristics of pyrethroid resistance in the malaria vector Anopheles funestus. Med Vet Entomol. 2005;19(3):271–5. doi: 10.1111/j.1365-2915.2005.00574.x Gillies MT, De Meillon B. The Anophelinae of Africa south of the Sahara (Ethiopian zoogeographical region). [Internet]. 1968 [cited 2024 Oct 15]. Available from: https://www.cabidigitallibrary.org/doi/full/ 10.5555/19692900946 Vezenegho SB, Chiphwanya J, Hunt RH, Coetzee M, Bass C, Koekemoer LL. Characterization of the Anopheles funestus group, including Anopheles funestus-like, from Northern Malawi. Trans R Soc Trop Med Hyg. 2013;107(12):753–62. doi: 10.1093/trstmh/trt089 PubMed PMID: 24189481. Dia I, Guelbeogo MW, Ayala D, Dia I, Guelbeogo MW, Ayala D. Advances and Perspectives in the Study of the Malaria Mosquito Anopheles funestus. In: Anopheles mosquitoes - New insights into malaria vectors [Internet]. IntechOpen; 2013 [cited 2025 Feb 7]. Available from: https://www.intechopen.com/chapters/43973 doi: 10.5772/55389 Ngowo HS, Hape EE, Matthiopoulos J, Ferguson HM, Okumu FO. Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization. Malar J. 2021;20(1):148. doi: 10.1186/s12936-021-03677-3 Kaindoa EW, Matowo NS, Ngowo HS, Mkandawile G, Mmbando A, Finda M, et al. Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south–eastern Tanzania. PLOS ONE. 2017;12(5):e0177807. doi: 10.1371/journal.pone.0177807 Lwetoijera DW, Harris C, Kiware SS, Dongus S, Devine GJ, McCall PJ, et al. Increasing role of Anopheles funestus and Anopheles arabiensis in malaria transmission in the Kilombero Valley, Tanzania. Malar J. 2014;13(1):331. doi: 10.1186/1475-2875-13-331 Ranson H, Lissenden N. Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016;Special Issue: Vectors32(3):187–96. doi: 10.1016/j.pt.2015.11.010 Coetzee M, Craig M, Sueur D le, Coetzee M, Craig M, Sueur D le. Distribution of African Malaria Mosquitoes Belonging to the Anopheles gambiae Complex. Parasitol Today. 2000;16(2):74–7. doi: 10.1016/S0169-4758 (99)01563-X PubMed PMID: 10652493. Mulamba C, Riveron JM, Ibrahim SS, Irving H, Barnes KG, Mukwaya LG, et al. Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms. PLOS ONE. 2014;9(10):e110058. doi: 10.1371/journal.pone.0110058 Riveron JM, Yunta C, Ibrahim SS, Djouaka R, Irving H, Menze BD, et al. A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector. Genome Biol. 2014;15(2):R27. doi: 10.1186/gb-2014-15-2-r27 PubMed PMID: 24565444; PubMed Central PMCID: PMC4054843. Cohuet A, Simard F, Toto JC, Kengne P, Coetzee M, Fontenille D. SPECIES IDENTIFICATION WITHIN THE ANOPHELES FUNESTUS GROUP OF MALARIA VECTORS IN CAMEROON AND EVIDENCE FOR A NEW SPECIES. Am J Trop Med Hyg. 2003;69(2):200–5. doi: 10.4269/ajtmh.2003.69.200 Moiroux N, Gomez MB, Pennetier C, Elanga E, Djènontin A, Chandre F, et al. Changes in Anopheles funestus Biting Behavior Following Universal Coverage of Long-Lasting Insecticidal Nets in Benin. J Infect Dis. 2012;206(10):1622–9. doi: 10.1093/infdis/jis565 Dida GO, Anyona DN, Abuom PO, Akoko D, Adoka SO, Matano AS, et al. Spatial distribution and habitat characterization of mosquito species during the dry season along the Mara River and its tributaries, in Kenya and Tanzania. Infect Dis Poverty. 2018;7(1):2. doi: 10.1186/s40249-017-0385-0 PubMed PMID: 29343279; PubMed Central PMCID: PMC5772712. Puchot N, Lecoq MT, Carinci R, Duchemin JB, Gendrin M, Bourgouin C. Establishment of a colony of Anopheles darlingi from French Guiana for vector competence studies on malaria transmission. Front Trop Dis. 2022;3. Located at: Scopus. doi: 10.3389/fitd.2022.949300 Giglio NF, Sousa-Lima AS, Gallardo AKR, Lima JBP. Laboratory Colonization of Anopheles (Nyssorhynchus) marajoara (Diptera: Culicidae) by Induced Copulation. J Med Entomol. 2015;52(1):3–8. doi: 10.1093/jme/tju004 Mrosso PC, Burke AM, Ngowo HS, Riddin MA, Okumu FO, Coetzee BWT, et al. Visual cues enhance mating success in laboratory colonies of the malaria vector Anopheles funestus with strain-specific responses [Internet]. 2025 [cited 2025 Jun 29]. Available from: https://www.researchsquare.com/article/rs-6527720/v1 doi: 10.21203/rs.3.rs-6527720/v1 Hape EE, Ngonyani AT, Mabula DM, Nkya JD, Thomas CA, Omari MJ, et al. Delayed mating in the malaria vector Anopheles funestus compared to Anopheles arabiensis (Diptera: Culicidae). J Med Entomol. 2025;62(4):921–9. doi: 10.1093/jme/tjaf059 Service MW, Oguamah D. Colonization of Anopheles funestus. Nature. 1958;181(4617):4617. doi: 10.1038/1811225b0 Niain’ny Felamboahangy L, Kaiser ML, Zengenene MP, Okumu F, Munhenga G, Koekemoer LL. Optimisation of laboratory-rearing parameters for Anopheles funestus larvae and adults. Acta Trop. 2023;238:106785. doi: 10.1016/j.actatropica.2022.106785 Zongo ON, Kiendrebeogo E, Sow BBD, Kientega M, Toé I, Sanou R, et al. Spatial Distribution and Biodiversity of Anopheles Mosquito Species Across Climatic Zones in Burkina Faso: Implications for Malaria Vector Control. Trop Med Infect Dis. 2025;11(1). doi: 10.3390/tropicalmed11010001 Dabiré KR, Baldet T, Diabaté A, Dia I, Carlo Costantini, Carlo Costantini, et al. Anopheles funestus (Diptera: Culicidae) in a Humid Savannah Area of Western Burkina Faso: Bionomics, Insecticide Resistance Status, and Role in Malaria Transmission. J Med Entomol. 2007;44(6):990–7. doi: 10.1603/0022-2585 (2007)44[990:afdcia]2.0.co;2 PubMed PMID: 18047197. Gillies MT, Coetzee M. A supplement to the Anophelinae of Africa South of the Sahara. Publ Afr Inst Med Res. 1987;55:1–143. Benedict MQ, Knols BGJ, Bossin HC, Howell PI, Mialhe E, Caceres C, et al. Colonisation and mass rearing: Learning from others. Malar J. 2009;8(SUPPL. 2). Located at: Scopus. doi: 10.1186/1475-2875-8-S2-S4 Koekemoer L, Kamau L, Hunt R, Coetzee M. Cocktail polymerase chain reaction assay to identify members of the Anopheles funestus (Diptera: Culicidae) group. Am J Trop Med Hyg. 2002;66:804–11. doi: 10.4269/ajtmh.2002.66.804 Morgan JC, Irving H, Okedi LM, Steven A, Wondji CS. Pyrethroid Resistance in an Anopheles funestus Population from Uganda. PLOS ONE. 2010;5(7):e11872. doi: 10.1371/journal.pone.0011872 Ngowo HS, Hape EE, Matthiopoulos J, Ferguson HM, Okumu FO. Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization. Malar J. 2021;20(1):148. doi: 10.1186/s12936-021-03677-3 Benedict MQ. yumpu.com [Internet]. [cited 2026 Mar 2]. Methods in Anopheles Research - MR4. Available from: https://www.yumpu.com/en/document/view/49409322/methods-in-anopheles-research-mr4 Guelbeogo WM, Sagnon N, Grushko O, Yameogo MA, Boccolini D, Besansky NJ, et al. Seasonal distribution of Anopheles funestus chromosomal forms from Burkina Faso. Malar J. 2009;8(1):239. doi: 10.1186/1475-2875-8-239 Hape EE, Ngonyani AT, Mabula DM, Nkya JD, Thomas CA, Omari MJ, et al. Delayed mating in the malaria vector Anopheles funestus compared to Anopheles arabiensis (Diptera: Culicidae). J Med Entomol. 2025;62(4):921–9. doi: 10.1093/jme/tjaf059 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 29 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 05 Apr, 2026 Editor assigned by journal 03 Apr, 2026 Submission checks completed at journal 03 Apr, 2026 First submitted to journal 01 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9291181","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620145641,"identity":"09adfc1c-f8e1-4115-9616-89883312f6ff","order_by":0,"name":"Grégoire Sawadogo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYFCDA0D8ocIGSDI2HiBaC+OMM2kgLQ3Ea2HmbTsMtxEnkI8+fPjlj5rD8nzH2y8+5m07b7e2/TDQlhqbaFxaDM+lpVnzHDtsOPPMmWLDOeduJ287kwjUciwttwGXlh4eM2MGttuMG27kpEm8KbudbHYAqIWx4TAeLfzfDH/8u20P1sLDdi7Z7PxD/FrkeXiYH/C23U7ccCP9mCRP2wE7sxsEbDHgYTNj5u37nwz0C7PhjDPJCWY3gLYk4PGLfA/z448/vqXZ9h1vf/jgQ4Wdvdn5dCCjxga3LQcY2CQgTB4DEJkIVpmAQznYlgYG5g8QJvsDEGmPR/EoGAWjYBSMUAAAovhvdE2RMCAAAAAASUVORK5CYII=","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":true,"prefix":"","firstName":"Grégoire","middleName":"","lastName":"Sawadogo","suffix":""},{"id":620145642,"identity":"cd1aa797-00b0-4b77-b311-5273c784c5a2","order_by":1,"name":"Saberé O.G. Yemien","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Saberé","middleName":"O.G.","lastName":"Yemien","suffix":""},{"id":620145643,"identity":"30cc34fa-729c-413a-a6c9-750f87c0f17c","order_by":2,"name":"Ibrahim Diallo","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Diallo","suffix":""},{"id":620145644,"identity":"6cfd9d75-458b-4677-b341-87631bdac546","order_by":3,"name":"Odette N. Zongo","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Odette","middleName":"N.","lastName":"Zongo","suffix":""},{"id":620145645,"identity":"87804f77-04b4-462f-ac7a-ee0fd3495989","order_by":4,"name":"Honorine Kaboré","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Honorine","middleName":"","lastName":"Kaboré","suffix":""},{"id":620145646,"identity":"806b891d-9965-4dc7-8da4-4a0a1f452112","order_by":5,"name":"Achaz A. M. Agolinou","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Achaz","middleName":"A. M.","lastName":"Agolinou","suffix":""},{"id":620145647,"identity":"eeafe0b6-849d-476c-8d6a-d7aa40bfdc47","order_by":6,"name":"Blandine Ouédraogo","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Blandine","middleName":"","lastName":"Ouédraogo","suffix":""},{"id":620145648,"identity":"b90f2792-83f1-43af-872b-f2cf226b11db","order_by":7,"name":"Stellah A Chumbe","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Stellah","middleName":"A","lastName":"Chumbe","suffix":""},{"id":620145649,"identity":"c4e8aa85-7eed-4eb2-bd8f-78fb07893232","order_by":8,"name":"Nouhoun Traoré","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Nouhoun","middleName":"","lastName":"Traoré","suffix":""},{"id":620145650,"identity":"727aca04-1298-4133-83c9-bd8794a1b0a2","order_by":9,"name":"Holly Humphreys","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Holly","middleName":"","lastName":"Humphreys","suffix":""},{"id":620145651,"identity":"a6399cbf-580d-487c-b299-31dc2e5f55ba","order_by":10,"name":"Seni Ilboudo","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Seni","middleName":"","lastName":"Ilboudo","suffix":""},{"id":620145652,"identity":"0a8a672e-b6a8-49e7-af53-b7669f87f914","order_by":11,"name":"Moussa Namountougou","email":"","orcid":"","institution":"Nazi Boni University","correspondingAuthor":false,"prefix":"","firstName":"Moussa","middleName":"","lastName":"Namountougou","suffix":""},{"id":620145653,"identity":"502fe410-64e0-42fa-9812-70f80e32e8a4","order_by":12,"name":"Mahamadi Kientega","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Mahamadi","middleName":"","lastName":"Kientega","suffix":""},{"id":620145654,"identity":"edc33fde-feb8-4b50-81f0-95332dedbe5b","order_by":13,"name":"Alessandra Lanfrancotti","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Lanfrancotti","suffix":""},{"id":620145655,"identity":"ed5d56d6-b638-4ba7-965b-94d358b55aaf","order_by":14,"name":"Tony Nolan","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tony","middleName":"","lastName":"Nolan","suffix":""},{"id":620145656,"identity":"c80652da-9ba1-4a58-ab44-32dbdb74d3d3","order_by":15,"name":"Hamidou Maiga","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Hamidou","middleName":"","lastName":"Maiga","suffix":""},{"id":620145657,"identity":"cb78868c-36af-4a8a-aba5-671309380908","order_by":16,"name":"Abdoulaye Diabaté","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé","correspondingAuthor":false,"prefix":"","firstName":"Abdoulaye","middleName":"","lastName":"Diabaté","suffix":""}],"badges":[],"createdAt":"2026-04-01 11:24:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9291181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9291181/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106644400,"identity":"a9e2ea63-9e02-4c52-915c-2be94284fdec","added_by":"auto","created_at":"2026-04-10 19:32:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54333,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic location of field sample collection sites\u003c/p\u003e\n\u003cp\u003eAdult \u003cem\u003eAnopheles funestus\u003c/em\u003e females used for colony establishment were collected in Soumousso and Diébougou. Bobo-Dioulasso indicates the location of the insectary where mosquitoes were maintained, and colonisation experiments were conducted.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/56ed412c2ecd5f0090eaeeab.jpg"},{"id":106727624,"identity":"1bb07a5a-dae3-492c-b2fc-8c225a0b0be0","added_by":"auto","created_at":"2026-04-12 18:39:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248148,"visible":true,"origin":"","legend":"\u003cp\u003eAdult mosquito collection\u003c/p\u003e\n\u003cp\u003eAdult mosquitoes were collected using mouth aspirators inside uninhabited and inhabited houses (\u003cstrong\u003eA\u003c/strong\u003eand \u003cstrong\u003eB\u003c/strong\u003e). In some cases, mosquitoes were also collected in peri-domestic water storage containers (\u003cstrong\u003eC\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/80d475a6485d6cfe228027b6.jpg"},{"id":106644402,"identity":"2a5acce2-1786-4eed-89f4-132256845e39","added_by":"auto","created_at":"2026-04-10 19:32:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16538,"visible":true,"origin":"","legend":"\u003cp\u003eFemale’s individual oviposition cups.\u003c/p\u003e\n\u003cp\u003eFemales were placed individually in cups covered with a fine mosquito net secured with an elastic band. Each cup was lined on the inner wall with a strip of filter paper to provide a suitable substrate for egg deposition. Approximately 50 mL of dechlorinated water was added to each cup to stimulate oviposition. Cups were monitored daily for oviposition.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/da27d76ccaa922dcd5f7a1f3.jpg"},{"id":106644403,"identity":"eb43faa7-131e-40f2-90c0-43af1ad8ec61","added_by":"auto","created_at":"2026-04-10 19:32:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":115848,"visible":true,"origin":"","legend":"\u003cp\u003eWorkflow for the establishment of an Anopheles funestuslaboratory colony.\u003c/p\u003e\n\u003cp\u003eMosquitoes were collected in the field using aspirators (1), transported alive to the insectary (2) and morphologically identified under a stereomicroscope (2). Individual oviposition assays were conducted to obtain single-family egg batches (4). Species identity of parental females was confirmed by PCR (5), and only Anopheles funestus s.s lines were retained (6). Larvae from confirmed females were pooled and reared to adulthood (7), enabling colony establishment and maintenance across generations (8).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/0e1ae3a0823888a08088d945.jpg"},{"id":106993582,"identity":"93acc68d-384d-49f0-aa18-a65be3991c1f","added_by":"auto","created_at":"2026-04-15 14:38:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39801,"visible":true,"origin":"","legend":"\u003cp\u003eColony maintenance workflow.\u003c/p\u003e\n\u003cp\u003eMosquitoes were blood-fed at 4 and 6–8 days post-emergence (1). Eggs bowls were placed 48 hours post blood-feeding and eggs were collected after 72 hours (2), hatched within 2 days (3), and larvae developed into pupae in about 2 weeks (4). Adults emerged 48 hours later (5), marking the start of a new cycle.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/10fea2561b8a58a604fe3d1a.jpg"},{"id":106726618,"identity":"faedfd7b-391d-4605-86ec-66de61fe4130","added_by":"auto","created_at":"2026-04-12 18:36:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1065988,"visible":true,"origin":"","legend":"\u003cp\u003eF0 female reproductive performance.\u003c/p\u003e\n\u003cp\u003e(A) number of eggs laid per female and (B) hatching rate per female. Each point represents an individual female; horizontal bars indicate the mean ± standard error.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/03bd5237050f866695ed7264.jpg"},{"id":106996182,"identity":"dd1e16e8-5753-4afa-903c-04da52b0bc5d","added_by":"auto","created_at":"2026-04-15 15:27:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2489131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9291181/v1/b67af0c4-69de-4a35-8375-23b3dff9735d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From the field to the laboratory: establishment of Anopheles funestus s.s. colony in Burkina Faso","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria remains a major global health concern, with an estimated 247\u0026nbsp;million cases worldwide and nearly half of the global population at risk [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In sub-Saharan Africa, four dominant mosquito species (\u003cem\u003eAn. gambiae s.s., An. arabiensis, An. coluzzii, and An. funestus s.s\u003c/em\u003e) drive malaria transmission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While extensive research has focused on the \u003cem\u003eAn. gambiae\u003c/em\u003e complex, \u003cem\u003eAn. funestus\u003c/em\u003e remains comparatively understudied, largely because it is exceptionally difficult to rear and maintain under laboratory conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAn. funestus\u003c/em\u003e species complex comprises at least 13 recognized members, including \u003cem\u003eAn. aruni\u003c/em\u003e, \u003cem\u003eAn. brucei\u003c/em\u003e, \u003cem\u003eAn. confusus\u003c/em\u003e, \u003cem\u003eAn. funestus\u003c/em\u003e sensu stricto (s.s.), \u003cem\u003eAn. funestus\u003c/em\u003e-like, \u003cem\u003eAn. fuscivenosus\u003c/em\u003e, \u003cem\u003eAn. leesoni\u003c/em\u003e, \u003cem\u003eAn. longipalpis\u003c/em\u003e type A, \u003cem\u003eAn. longipalpis\u003c/em\u003e type C, \u003cem\u003eAn. parensis\u003c/em\u003e, \u003cem\u003eAn. rivulorum\u003c/em\u003e, \u003cem\u003eAn. rivulorum-like\u003c/em\u003e, and \u003cem\u003eAn. vaneedeni\u003c/em\u003e [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The vectorial capacity of these species varies, and only \u003cem\u003eAn. funestus\u003c/em\u003e s.s. contribute to the spread of malaria parasite in certain regions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eAn. funestus\u003c/em\u003e s.s control is mainly based on the use of insecticide through impregnated nets. Unfortunately, the effectiveness of this control strategy has been compromised for different reasons [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This species exhibits strong anthropophilic and endophilic behaviour, high levels of insecticide resistance, and longer adult longevity than other major African malaria vectors [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These characteristics make it a highly efficient vector and an important driver of persistent malaria transmission, particularly in areas where control programmes depend largely on insecticide-based interventions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nevertheless, control failure in \u003cem\u003eAn. funestus\u003c/em\u003e is unlikely to be explained by insecticide resistance alone. Behavioural plasticity, including exophilic, exophagic, and partially zoophilic tendencies reported in some populations, may allow this species to evade contact with long-lasting insecticidal nets and indoor residual spraying [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, because \u003cem\u003eAn. funestus\u003c/em\u003e often reaches high densities later in the transmission season and persists into the dry season through its exploitation of more permanent, vegetated breeding sites, its peak abundance may not fully coincide with the timing of control efforts that are often designed around the rainy-season dynamics of \u003cem\u003eAn. gambiae\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These combined behavioural, ecological, and physiological features likely contribute to the limited effectiveness of standard interventions against \u003cem\u003eAn. funestus\u003c/em\u003e and to its continuing role in residual malaria transmission.\u003c/p\u003e \u003cp\u003eThe establishment of laboratory mosquito colonies has been pivotal in advancing understanding of their biology, including developmental processes, mechanisms of insecticide resistance, and pathogen transmission dynamics. Such colonies also provide standardised conditions for evaluating novel insecticides and innovative vector control strategies, including gene drive tools [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mosquito colonisation remains challenging, particularly for eurygamic species such as \u003cem\u003eAnopheles funestus\u003c/em\u003e, which show limited mating success in confined laboratory conditions, low oviposition rates, and high sensitivity to environmental fluctuations such as temperature and humidity[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAttempts to colonise \u003cem\u003eAn. funestus\u003c/em\u003e dates back to the 1950s, when an initial effort in Nigeria failed after only a few months [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To date, only a few laboratory colonies have been successfully maintained for extended generations, including: the FANG strain (Funestus ANGola), the FUMOZ strain (Funestus MOZambique) and more recently the FUTAZ from Tanzania [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Given the broad geographic distribution of \u003cem\u003eAnopheles funestus\u003c/em\u003e sensu stricto (a member of the \u003cem\u003eAn. funestus\u003c/em\u003e species complex) and the diversity of ecological and operational environments in which it occurs, significant genetic structuring may exist among populations. This variability may be shaped by local ecological adaptation, insecticide selection pressure, and levels of gene flow, resulting in differences in key vectorial traits across regions. It is therefore important to distinguish variation among geographically distinct populations of \u003cem\u003eAn. funestus\u003c/em\u003e s.s. from differences attributable to other, morphologically similar species within the complex. As a result, reliance on a single laboratory strain may not adequately represent the biological and epidemiological diversity of the species. The establishment of stable laboratory colonies from multiple geographic origins is therefore essential to support robust investigations of \u003cem\u003eAn. funestus\u003c/em\u003e biology, insecticide resistance, parasite\u0026ndash;vector interactions, and the assessment of novel vector control strategies, including genetic control methods.\u003c/p\u003e \u003cp\u003eIn this study, we describe the establishment of the first \u003cem\u003eAnopheles funestus\u003c/em\u003e laboratory colony from West Africa, derived from field populations collected in Burkina Faso. This represents an important advance, given that \u003cem\u003eAn. funestus\u003c/em\u003e is a major malaria vector in West Africa and that no colony from this region has previously been available. We detail the collection methods and colony maintenance protocols implemented to support successful establishment. By documenting these procedures, we provide practical guidance for laboratories seeking to colonise \u003cem\u003eAn. funestus\u003c/em\u003e, thereby strengthening research capacity on this ecologically and epidemiologically important vector and supporting future studies, including those aimed at developing locally relevant control strategies.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito field collection sites\u003c/h2\u003e \u003cp\u003eFollowing our countrywide entomological surveillance in 2022 across about 70 districts, a few sites were identified as conducive for \u003cem\u003eAn. funestus\u003c/em\u003e occurrence [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, \u003cem\u003eAnopheles\u003c/em\u003e adult mosquitoes were collected from two localities in western Burkina Faso, including Di\u0026eacute;bougou (10\u0026deg;57\u0026rsquo;41\u0026rdquo; N, 3\u0026deg;15\u0026rsquo;00\u0026rdquo; W) and Soumousso (11\u0026deg;00\u0026rsquo;46\u0026rdquo; N, 4\u0026deg;02\u0026rsquo;45\u0026rdquo; W) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDi\u0026eacute;bougou is located 136 km south-east of Bobo-Dioulasso, the second-largest city in Burkina Faso. In these sites, several \u003cem\u003eAnopheles\u003c/em\u003e species coexist, including \u003cem\u003eAn. gambiae\u003c/em\u003e s.s, \u003cem\u003eAn. coluzzii\u003c/em\u003e and \u003cem\u003eAn. funestus\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Previous studies have reported high densities of \u003cem\u003eAn. funestus\u003c/em\u003e toward the end of the rainy season and during the cool dry period [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These conditions make the area particularly suitable for sampling and for establishing \u003cem\u003eAn. funestus\u003c/em\u003e laboratory colonies.\u003c/p\u003e \u003cp\u003eSoumousso is a typical village in the Guinean savannah, located 55 km east of Bobo-Dioulasso. The village is home to the primary malaria vectors: \u003cem\u003eAn. coluzzii\u003c/em\u003e, \u003cem\u003eAn. gambiae s.s, An. funestus\u003c/em\u003e, and \u003cem\u003eAn. nili\u003c/em\u003e. Additionally, \u003cem\u003eAn. arabiensis\u003c/em\u003e is occasionally found at low frequency, accounting for approximately 5% of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. samples [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdult \u003cem\u003eAnopheles funestus\u003c/em\u003e females used for colony establishment were collected in Soumousso and Di\u0026eacute;bougou. Bobo-Dioulasso indicates the location of the insectary where mosquitoes were maintained, and colonisation experiments were conducted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMosquito collection period and handling\u003c/h3\u003e\n\u003cp\u003eAdult female mosquitoes were collected at the end of the rainy season (October to December) in 2023 and 2025 between 5 a.m. and 7 a.m. Mosquitoes were sampled both indoors and outdoors from inhabited and uninhabited houses and chicken coops, as well as from resting sites such as clay water jars and tree holes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To maximise capture efficiency while preserving specimen viability, both mouth aspirators and battery-powered electric aspirators were used. The latter was operated at reduced voltage to reduce suction force, thereby minimising injury or mortality during mosquito collection. Mosquitoes were collected in cages measuring 30cm x 30cm x 30 cm. Once collection was complete, the cages were covered with wet mops and transported in an air-conditioned vehicle to the laboratory. All collected mosquitoes were morphologically identified to genus and species level using the identification keys of Gillies and Coetzee [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] then transported to the insectary at the Institut de Recherche en Sciences de la Sant\u0026eacute; (IRSS) for individual oviposition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdult mosquitoes were collected using mouth aspirators inside uninhabited and inhabited houses (\u003cb\u003eA\u003c/b\u003e and \u003cb\u003eB\u003c/b\u003e). In some cases, mosquitoes were also collected in peri-domestic water storage containers (\u003cb\u003eC\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnopheles funestus\u003c/b\u003e \u003cb\u003estrain establishment\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eInsectary conditions\u003c/h3\u003e\n\u003cp\u003eAdult mosquitoes were maintained under standard insectary conditions at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and 75\u0026ndash;80% relative humidity, the larvae were kept at 29\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with a 12:12 h light-dark cycle [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIndividual oviposition\u003c/h3\u003e\n\u003cp\u003eIndividual female mosquitoes were isolated for oviposition in 200 mL disposable cups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Each cup was lined with a strip of filter paper (15 \u0026times; 5 cm) to provide a suitable substrate for egg deposition. Approximately 50 mL of deionised water was added to each cup to stimulate oviposition. The cups were covered with a fine mosquito net secured with an elastic band, incorporating a small central slit to allow careful introduction of females without injury. Each cup was assigned a unique identification code to enable accurate tracking of individual females and their progeny. A cotton pad soaked in a 10% glucose solution was placed on top of the netting and replenished daily to maintain adult survival during the oviposition period. All cups were kept under insectary conditions and were inspected daily to assess oviposition success. Females that laid eggs were removed and preserved individually in 1.5 mL Eppendorf tubes containing 80% ethanol, with tube labels corresponding to the unique identifiers of their respective cups. Eggs were carefully transferred to larval trays containing deionised water and maintained under insectary conditions until hatching. Larvae of \u003cem\u003eAn. funestus\u003c/em\u003e s.s PCR positive were pooled and reared to adulthood with finely ground TetraMin\u0026reg; fish food.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFemales were placed individually in cups covered with a fine mosquito net secured with an elastic band. Each cup was lined on the inner wall with a strip of filter paper to provide a suitable substrate for egg deposition. Approximately 50 mL of dechlorinated water was added to each cup to stimulate oviposition. Cups were monitored daily for oviposition.\u003c/p\u003e\n\u003ch3\u003eMolecular identification of founders\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from single mosquitoes using DNazol protocol and analysed by multiplex Polymerase Chain Reaction (PCR) assay [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to distinguish sibling species within the \u003cem\u003eAn. funestus\u003c/em\u003e group. PCR reactions were carried out in a final volume of 20 \u0026micro;l, consisting of 4 \u0026micro;l of 5X FIREPol\u0026reg; Master Mix,11.9 \u0026micro;l of nuclease-free water, 0.3 \u0026micro;l of each of the five species-specific primers, and 2 \u0026micro;l of genomic DNA template. Thermal cycling conditions consisted of an: initial denaturation at 94\u0026deg;C for 10 min; 35 cycles of denaturation at 94\u0026deg;C for 45 s, annealing at 45\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 40 s; followed by a final extension at 72\u0026deg;C for 10 min [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. PCR products were resolved by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualised under UV illumination. Amplicon sizes were compared against a 100bp molecular size marker and existing positive controls of \u003cem\u003eAn. funestus s.s\u003c/em\u003e to determine species identity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMosquitoes were collected in the field using aspirators (1), transported alive to the insectary (2) and morphologically identified under a stereomicroscope (2). Individual oviposition assays were conducted to obtain single-family egg batches (4). Species identity of parental females was confirmed by PCR (5), and only \u003cem\u003eAnopheles funestus s.s\u003c/em\u003e lines were retained (6). Larvae from confirmed females were pooled and reared to adulthood (7), enabling colony establishment and maintenance across generations (8).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony maintenance and challenges beyond the F1 generation\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eLarval and adult feeding regimes\u003c/h2\u003e \u003cp\u003eLarvae from the F1 generation onwards were maintained under standard insectary conditions (see Insectary conditions section above) in rectangular trays (30 cm \u0026times; 24 cm \u0026times; 6 cm) containing 1 L of deionised water. Finely ground TetraMin\u0026reg; fish food was provided as the larval diet (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Adults were maintained on a 10% glucose solution provided \u003cem\u003ead libitum\u003c/em\u003e, with a cotton pad soaked daily.\u003c/p\u003e \u003cp\u003eThe first blood-feeding occurred four days after the last adult emergence. The interval between the first and second blood-feeding ranged from two to four days. During colony establishment, blood-feeding was attempted at 2:00 pm, 4:00 pm, 6:00 pm, 7:00 pm, and 9:00 pm using the Hemotek system with rabbit blood. As these preliminary observations were not part of a formal experiment, no comparative analysis was performed; 7:00 pm was selected for subsequent blood-feeding. Mosquitoes were starved for 5 hours before each blood feed.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLarvae feeding schedule for 250 larvae in a tray\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarval age\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg per larva\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003emg per tray\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFeeding schedule\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 0 (Floating)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay1 (splitting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 drops\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 drops\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 drop + \u0026frac12; drop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e1 drop\u0026thinsp;=\u0026thinsp;30mg\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eReproductive performance and oviposition challenges\u003c/h3\u003e\n\u003cp\u003eA major difficulty in colony maintenance was that \u003cem\u003eAn. funestus\u003c/em\u003e females did not readily lay eggs under standard insectary conditions. To stimulate oviposition, several strategies were tested to reproduce natural breeding cues. These included providing moistened cotton pads in Petri dishes, placing bowls filled with tap water, filtered larval water, and, in some cases, adding fresh grass stems to the bowls.\u003c/p\u003e \u003cp\u003eDuring routine colony management two consecutive blood meals were required. Four days after the second blood meal, oviposition bowls were placed in the adult cages. Each bowl contained deionised water with filter paper partially immersed along the sides and supplemented with a small quantity of fresh grass. Eggs were then transferred to trays and larvae were maintained on ground TetraMin\u0026reg; with feeding quantities adjusted according to larval developmental stage and their quantity (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMosquitoes were blood-fed at 4 and 6\u0026ndash;8 days post-emergence (1). Eggs bowls were placed 48 hours post blood-feeding and eggs were collected after 72 hours (2), hatched within 2 days (3), and larvae developed into pupae in about 2 weeks (4). Adults emerged 48 hours later (5), marking the start of a new cycle.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFecundity and fertility assessment\u003c/h2\u003e \u003cp\u003eTo assess \u003cem\u003eAn. funestus\u003c/em\u003e fecundity and fertility in laboratory condition, females were allowed to individually lay eggs in oviposition cups. Among the females that successfully laid eggs, 48 were randomly selected for fecundity and hatching assessment. The total number of eggs laid by each female was counted under a stereomicroscope. Oviposition cups were inspected daily, and upon hatching, first-instar larvae were counted and transferred to a new rearing container. The original oviposition cups were subsequently monitored for an additional three days to record any delayed hatching.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eWild-caught mosquitoes for colony establishment\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAdult mosquitoes were collected from October to December 2023 and 2025 in Soumousso and Diebougou to support the establishment of \u003cem\u003eAn. funestus\u003c/em\u003e laboratory strain in the insectary. Female mosquitoes were collected alive at each site, transferred to cages (30cm \u0026times; 30cm \u0026times; 30cm), fed with a 10% glucose solution and transported to the laboratory for colony establishment. A total of 1,871 mosquitoes were morphologically identified as \u003cem\u003eAn. funestus s.l\u003c/em\u003e. Of these, 794 females laid eggs, corresponding to a 42.44% success rate across collections (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of \u003cem\u003eAnopheles funestus\u003c/em\u003e collections and oviposition outcomes by collection year\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollection_years\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber_collected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal_laid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal_unlaid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%Laid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%Unlaid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1,111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e67.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1,871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e57.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eColony maintenance\u003c/h2\u003e \u003cp\u003eTo ensure successful colony maintenance, several procedural adjustments were implemented. Blood feeding was standardized at 7:00 pm and performed twice prior to oviposition. In addition, oviposition success improved when females were provided with bowls lined with filter paper and supplemented with fresh grass, which appeared to stimulate egg laying.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFecundity and hatching success of founder\u003c/b\u003e \u003cb\u003eAn. funestus\u003c/b\u003e \u003cb\u003efemales\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale reproductive performance showed variability across individuals. The number of eggs laid per female ranged broadly, with a mean fecundity of 85\u0026plusmn; 2.9 eggs per female. Despite this variability in egg production, fertility remained high, as hatching rates were consistently elevated across females, with most individuals showing hatching success above 80%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) number of eggs laid per female and (B) hatching rate per female. Each point represents an individual female; horizontal bars indicate the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe establishment of laboratory colonies of malaria vectors is fundamental to research on their biology, behaviour, and control. Such colonies enable consistent, controlled evaluation of insecticides and novel control strategies, while also providing insight into variation in vector susceptibility and resistance mechanisms. In this context, successful colonisation represents an important step toward generating reproducible experimental material for studies that are difficult to conduct using field-caught mosquitoes alone.\u003c/p\u003e \u003cp\u003eThis study demonstrates the feasibility of establishing a local colony of \u003cem\u003eAn. funestus s.s.\u003c/em\u003e in Burkina Faso, a species recognised as difficult to maintain in captivity due to its sensitivity to rearing conditions, and low egg-laying rate in the laboratory [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The colony is successfully maintained up to generation 23, illustrating the effectiveness of the strategies implemented to optimise collection, egg laying, and rearing.\u003c/p\u003e \u003cp\u003eField collection methods were specifically adapted to maximize mosquito survival for colonization purposes. Mouth aspirators, although limited in throughput, allowed the collection of live females with minimal physical damage. In parallel, battery-powered mechanical aspirators were used at reduced suction power to increase collection efficiency while limiting injury-induced mortality. The combination of these approaches enabled the collection of more than 2,000 live specimens, with 1,871 individuals morphologically identified as belonging to \u003cem\u003eAn. funestus\u003c/em\u003e group.\u003c/p\u003e \u003cp\u003eTransport from field sites to the insectary represents a critical bottleneck in colony establishment, particularly for \u003cem\u003eAn. funestus\u003c/em\u003e, a species known to be sensitive to environmental stress[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Mortality during transport is commonly associated with desiccation and heat stress, especially under tropical field conditions. To mitigate these effects, transport cages were covered with wet mops to maintain humidity, and vehicle air conditioning was used to reduce thermal stress. Such measures are consistent with established recommendations for maintaining field-collected Anopheles under optimal survival conditions prior to insectary processing [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These operational adaptations were essential to preserve female viability and ensure successful downstream oviposition, thereby contributing to the overall colonization effort.\u003c/p\u003e \u003cp\u003eMolecular identification of ovipositing females revealed that the majority belonged to \u003cem\u003eAn funestus s.s\u003c/em\u003e in both sampling years. In 2023, 85.6% of egg-laying females were confirmed as \u003cem\u003eAn. funestus s.s\u003c/em\u003e., while in 2024 this proportion decreased to 77.8%. These findings indicate that although \u003cem\u003eAn. funestus s.s\u003c/em\u003e. was the predominant species among reproducing females, a non-negligible proportion of morphologically identified specimens belonged to other members of the \u003cem\u003eAn. funestus\u003c/em\u003e group.\u003c/p\u003e \u003cp\u003eSuccessful colonization of \u003cem\u003eAn. funestus\u003c/em\u003e has historically been constrained by its behavioral and ecological specificity, requiring careful optimisation of insectary conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, sustained colony maintenance was achieved after procedural adjustments targeting blood feeding and oviposition behavior. Standardizing blood feeding at 7:00 pm and providing two consecutive blood meals prior to oviposition likely aligned feeding with the species\u0026rsquo; natural nocturnal biting rhythm, thereby enhancing blood feeding rates and subsequent egg development. The strong association between circadian feeding patterns and reproductive output has been well documented in Anopheles mosquitoes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, oviposition success improved when females were provided with bowls lined with filter paper and supplemented with fresh grass. \u003cem\u003eAn. funestus\u003c/em\u003e is known to preferentially oviposit in permanent or semi-permanent water bodies with emergent vegetation, unlike species such as \u003cem\u003eAn. gambiae\u003c/em\u003e that exploit temporary habitats [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The incorporation of grass likely mimicked natural breeding-site cues, providing tactile or chemical stimuli that triggered egg laying. Similar substrate-dependent oviposition behavior has been reported in laboratory and semi-field studies of the An. funestus group [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEgg-laying rates of 42.44% combined with the high proportion of engorged females, suggest that mosquitoes acquire blood meals primarily within human dwellings before resting in nearby uninhabited structures. This resting behaviour highlights potential limitations of vector control strategies relying exclusively on indoor residual spraying. Similar patterns have been reported previously in the same area, particularly in Soumousso, where approximately 40% of \u003cem\u003eAn. funestus\u003c/em\u003e were collected outdoors compared to 60% indoors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], supporting the presence of flexible resting behaviour in this species.\u003c/p\u003e \u003cp\u003eBeyond these operational parameters, we assessed fecundity and fertility, two key indicators not only for establishing laboratory strains but also for implementing vector control strategies such as gene drives and the sterile insect technique. Our results showed a mean fecundity of 85\u0026plusmn;2.9 eggs per female and a mean hatching rate of 88.62%. These values are broadly comparable to those reported for the FUMOZ strain (76 eggs per female) and the recently established FUTAZ strain from Tanzania (64 eggs per female) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, it is important to note that in the present study, these parameters were evaluated using mosquitoes collected directly from the field (F0 generation). Differences in fecundity and fertility may therefore reflect ecological, physiological, or nutritional factors associated with field-collected females, as well as the absence of laboratory induced constraints observed in long-term colonised strains. Consequently, these results should be interpreted as indicative of the reproductive potential of field-derived \u003cem\u003eAn. funestus\u003c/em\u003e rather than as direct measures of long-term colony performance.\u003c/p\u003e \u003cp\u003eTaken together, these findings demonstrate that \u003cem\u003eAn. funestus\u003c/em\u003e populations from Burkina Faso exhibit high reproductive potential and behavioural flexibility, both of which are critical for successful early laboratory colonisation. The ability to obtain eggs beyond the F0 generation, combined with the high fecundity and hatching rates observed in field-derived females, highlights the feasibility of establishing a stable laboratory strain when key operational conditions are met. Although further optimisation and controlled evaluations across successive generations will be required, particularly to assess mating success and long-term colony performance, these results provide a practical framework for future colonisation efforts. Importantly, establishing a local \u003cem\u003eAn. funestus\u003c/em\u003e strain would support downstream applications, including insecticide resistance studies, behavioural assays, and the evaluation of novel vector control strategies.\u003c/p\u003e \u003cp\u003eAlthough our study was broadly successful, there were some limitations. Albeit the colony was established initially, several parameters used during colony maintenance, such as adding grasses in oviposition bowls to stimulate egg-laying and adjusting blood-feeding schedules, were applied empirically and may require further optimisation. Previous work has shown that oviposition cues, blood-feeding timing, and microenvironmental structure can strongly influence the reproductive performance of \u003cem\u003eAn. funestus\u003c/em\u003e and other eurygamic species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], suggesting that a more systematic evaluation of these factors will be essential to improve colony stability. Future studies should refine these conditions and explore alternative stimuli that better support consistent oviposition and successful progression to subsequent generations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study report successful colonisation of \u003cem\u003eAn. funestus\u003c/em\u003e in western Burkina Faso. Considering the inherent difficulty of establishing this species under laboratory conditions, our observations traced these factors back to field collection, revealing that \u003cem\u003eAn. funestus\u003c/em\u003e rest not only in inhabited houses but also abundantly in uninhabited houses. Colony maintenance required specific adjustments, notably blood feeding at 7:00 pm and the use of oviposition bowls containing fresh grasses. Further investigations are required to characterise this strain, particularly its insecticide resistance profile, and to optimise critical parameters such as larval nutrition, which remains essential for sustainable colony maintenance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have read and accepted this version of the manuscript. The authors also declare there are no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Bill \u0026amp; Melinda Gates Foundation (INV037164).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eG.S, S.O.G.Y, O.N.Z, M.K, H.K, A.A, N.T, I.D, H.M and AD conceived the study. G.S, S.O.G.Y, O.N.Z, H.K, A.A and S.I performed the field collection. G.S, S.O.G.Y, O.N.Z, S.C, B.O, A.A, H.K and I.D performed experiments. G.S performed the data analyses. G.S wrote the early version of the manuscript. A.D secured the funding acquisition. S.O.G.Y, O.N.Z, M.K, H.K; N.T, H.M, H.H, A.L, T.N and A.D edited and validated the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to acknowledge with appreciation the valuable collaboration and technical support provided by the Institut de Recherche en Sciences de la Sant\u0026eacute;, Direction R\u0026eacute;gionale de l\u0026rsquo;Ouest (IRSS-DRO, Burkina Faso).\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eData are provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. World malaria report 2023 [Internet]. 2023 [cited 2024 Aug 23]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023\u003c/span\u003e\u003cspan address=\"https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoetzee M, Koekemoer LL. Molecular systematics and insecticide resistance in the major African malaria vector Anopheles funestus. Annu Rev Entomol. 2013;58:393\u0026ndash;412. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-ento-120811-153628\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-120811-153628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e PubMed PMID: 23317045.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, et al. A global map of dominant malaria vectors. Parasit Vectors. 2012;5:69. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1756-3305-5-69\u003c/span\u003e\u003cspan address=\"10.1186/1756-3305-5-69\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e PubMed PMID: 22475528; PubMed Central PMCID: PMC3349467.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunt RH, Brooke BD, Pillay C, Koekemoer LL, Coetzee M. Laboratory selection for and characteristics of pyrethroid resistance in the malaria vector Anopheles funestus. Med Vet Entomol. 2005;19(3):271\u0026ndash;5. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2915.2005.00574.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2915.2005.00574.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGillies MT, De Meillon B. The Anophelinae of Africa south of the Sahara (Ethiopian zoogeographical region). [Internet]. 1968 [cited 2024 Oct 15]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cabidigitallibrary.org/doi/full/\u003c/span\u003e\u003cspan address=\"https://www.cabidigitallibrary.org/doi/full/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5555/19692900946\u003c/span\u003e\u003cspan address=\"10.5555/19692900946\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezenegho SB, Chiphwanya J, Hunt RH, Coetzee M, Bass C, Koekemoer LL. Characterization of the Anopheles funestus group, including Anopheles funestus-like, from Northern Malawi. Trans R Soc Trop Med Hyg. 2013;107(12):753\u0026ndash;62. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/trstmh/trt089\u003c/span\u003e\u003cspan address=\"10.1093/trstmh/trt089\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e PubMed PMID: 24189481.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDia I, Guelbeogo MW, Ayala D, Dia I, Guelbeogo MW, Ayala D. Advances and Perspectives in the Study of the Malaria Mosquito Anopheles funestus. In: Anopheles mosquitoes - New insights into malaria vectors [Internet]. IntechOpen; 2013 [cited 2025 Feb 7]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.intechopen.com/chapters/43973\u003c/span\u003e\u003cspan address=\"https://www.intechopen.com/chapters/43973\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5772/55389\u003c/span\u003e\u003cspan address=\"10.5772/55389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgowo HS, Hape EE, Matthiopoulos J, Ferguson HM, Okumu FO. Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization. Malar J. 2021;20(1):148. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12936-021-03677-3\u003c/span\u003e\u003cspan address=\"10.1186/s12936-021-03677-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaindoa EW, Matowo NS, Ngowo HS, Mkandawile G, Mmbando A, Finda M, et al. Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south\u0026ndash;eastern Tanzania. PLOS ONE. 2017;12(5):e0177807. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0177807\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0177807\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLwetoijera DW, Harris C, Kiware SS, Dongus S, Devine GJ, McCall PJ, et al. Increasing role of Anopheles funestus and Anopheles arabiensis in malaria transmission in the Kilombero Valley, Tanzania. Malar J. 2014;13(1):331. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1475-2875-13-331\u003c/span\u003e\u003cspan address=\"10.1186/1475-2875-13-331\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRanson H, Lissenden N. Insecticide Resistance in African \u003cem\u003eAnopheles\u003c/em\u003e Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016;Special Issue: Vectors32(3):187\u0026ndash;96. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.pt.2015.11.010\u003c/span\u003e\u003cspan address=\"10.1016/j.pt.2015.11.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoetzee M, Craig M, Sueur D le, Coetzee M, Craig M, Sueur D le. Distribution of African Malaria Mosquitoes Belonging to the Anopheles gambiae Complex. Parasitol Today. 2000;16(2):74\u0026ndash;7. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0169-4758\u003c/span\u003e\u003cspan address=\"10.1016/S0169-4758\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e(99)01563-X PubMed PMID: 10652493.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMulamba C, Riveron JM, Ibrahim SS, Irving H, Barnes KG, Mukwaya LG, et al. Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms. PLOS ONE. 2014;9(10):e110058. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0110058\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0110058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiveron JM, Yunta C, Ibrahim SS, Djouaka R, Irving H, Menze BD, et al. A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector. Genome Biol. 2014;15(2):R27. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/gb-2014-15-2-r27\u003c/span\u003e\u003cspan address=\"10.1186/gb-2014-15-2-r27\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e PubMed PMID: 24565444; PubMed Central PMCID: PMC4054843.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohuet A, Simard F, Toto JC, Kengne P, Coetzee M, Fontenille D. SPECIES IDENTIFICATION WITHIN THE ANOPHELES FUNESTUS GROUP OF MALARIA VECTORS IN CAMEROON AND EVIDENCE FOR A NEW SPECIES. Am J Trop Med Hyg. 2003;69(2):200\u0026ndash;5. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4269/ajtmh.2003.69.200\u003c/span\u003e\u003cspan address=\"10.4269/ajtmh.2003.69.200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoiroux N, Gomez MB, Pennetier C, Elanga E, Dj\u0026egrave;nontin A, Chandre F, et al. Changes in Anopheles funestus Biting Behavior Following Universal Coverage of Long-Lasting Insecticidal Nets in Benin. J Infect Dis. 2012;206(10):1622\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/infdis/jis565\u003c/span\u003e\u003cspan address=\"10.1093/infdis/jis565\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDida GO, Anyona DN, Abuom PO, Akoko D, Adoka SO, Matano AS, et al. Spatial distribution and habitat characterization of mosquito species during the dry season along the Mara River and its tributaries, in Kenya and Tanzania. Infect Dis Poverty. 2018;7(1):2. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40249-017-0385-0\u003c/span\u003e\u003cspan address=\"10.1186/s40249-017-0385-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e PubMed PMID: 29343279; PubMed Central PMCID: PMC5772712.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuchot N, Lecoq MT, Carinci R, Duchemin JB, Gendrin M, Bourgouin C. Establishment of a colony of Anopheles darlingi from French Guiana for vector competence studies on malaria transmission. Front Trop Dis. 2022;3. Located at: Scopus. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fitd.2022.949300\u003c/span\u003e\u003cspan address=\"10.3389/fitd.2022.949300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiglio NF, Sousa-Lima AS, Gallardo AKR, Lima JBP. Laboratory Colonization of Anopheles (Nyssorhynchus) marajoara (Diptera: Culicidae) by Induced Copulation. J Med Entomol. 2015;52(1):3\u0026ndash;8. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jme/tju004\u003c/span\u003e\u003cspan address=\"10.1093/jme/tju004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMrosso PC, Burke AM, Ngowo HS, Riddin MA, Okumu FO, Coetzee BWT, et al. Visual cues enhance mating success in laboratory colonies of the malaria vector Anopheles funestus with strain-specific responses [Internet]. 2025 [cited 2025 Jun 29]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchsquare.com/article/rs-6527720/v1\u003c/span\u003e\u003cspan address=\"https://www.researchsquare.com/article/rs-6527720/v1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.21203/rs.3.rs-6527720/v1\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-6527720/v1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHape EE, Ngonyani AT, Mabula DM, Nkya JD, Thomas CA, Omari MJ, et al. Delayed mating in the malaria vector Anopheles funestus compared to Anopheles arabiensis (Diptera: Culicidae). J Med Entomol. 2025;62(4):921\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jme/tjaf059\u003c/span\u003e\u003cspan address=\"10.1093/jme/tjaf059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eService MW, Oguamah D. Colonization of Anopheles funestus. Nature. 1958;181(4617):4617. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/1811225b0\u003c/span\u003e\u003cspan address=\"10.1038/1811225b0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiain\u0026rsquo;ny Felamboahangy L, Kaiser ML, Zengenene MP, Okumu F, Munhenga G, Koekemoer LL. Optimisation of laboratory-rearing parameters for Anopheles funestus larvae and adults. Acta Trop. 2023;238:106785. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.actatropica.2022.106785\u003c/span\u003e\u003cspan address=\"10.1016/j.actatropica.2022.106785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZongo ON, Kiendrebeogo E, Sow BBD, Kientega M, To\u0026eacute; I, Sanou R, et al. Spatial Distribution and Biodiversity of Anopheles Mosquito Species Across Climatic Zones in Burkina Faso: Implications for Malaria Vector Control. Trop Med Infect Dis. 2025;11(1). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/tropicalmed11010001\u003c/span\u003e\u003cspan address=\"10.3390/tropicalmed11010001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDabir\u0026eacute; KR, Baldet T, Diabat\u0026eacute; A, Dia I, Carlo Costantini, Carlo Costantini, et al. Anopheles funestus (Diptera: Culicidae) in a Humid Savannah Area of Western Burkina Faso: Bionomics, Insecticide Resistance Status, and Role in Malaria Transmission. J Med Entomol. 2007;44(6):990\u0026ndash;7. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1603/0022-2585\u003c/span\u003e\u003cspan address=\"10.1603/0022-2585\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e(2007)44[990:afdcia]2.0.co;2 PubMed PMID: 18047197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGillies MT, Coetzee M. A supplement to the Anophelinae of Africa South of the Sahara. Publ Afr Inst Med Res. 1987;55:1\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenedict MQ, Knols BGJ, Bossin HC, Howell PI, Mialhe E, Caceres C, et al. Colonisation and mass rearing: Learning from others. Malar J. 2009;8(SUPPL. 2). Located at: Scopus. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1475-2875-8-S2-S4\u003c/span\u003e\u003cspan address=\"10.1186/1475-2875-8-S2-S4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoekemoer L, Kamau L, Hunt R, Coetzee M. Cocktail polymerase chain reaction assay to identify members of the Anopheles funestus (Diptera: Culicidae) group. Am J Trop Med Hyg. 2002;66:804\u0026ndash;11. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4269/ajtmh.2002.66.804\u003c/span\u003e\u003cspan address=\"10.4269/ajtmh.2002.66.804\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan JC, Irving H, Okedi LM, Steven A, Wondji CS. Pyrethroid Resistance in an Anopheles funestus Population from Uganda. PLOS ONE. 2010;5(7):e11872. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0011872\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0011872\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgowo HS, Hape EE, Matthiopoulos J, Ferguson HM, Okumu FO. Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization. Malar J. 2021;20(1):148. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12936-021-03677-3\u003c/span\u003e\u003cspan address=\"10.1186/s12936-021-03677-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenedict MQ. yumpu.com [Internet]. [cited 2026 Mar 2]. Methods in Anopheles Research - MR4. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.yumpu.com/en/document/view/49409322/methods-in-anopheles-research-mr4\u003c/span\u003e\u003cspan address=\"https://www.yumpu.com/en/document/view/49409322/methods-in-anopheles-research-mr4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuelbeogo WM, Sagnon N, Grushko O, Yameogo MA, Boccolini D, Besansky NJ, et al. Seasonal distribution of Anopheles funestus chromosomal forms from Burkina Faso. Malar J. 2009;8(1):239. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1475-2875-8-239\u003c/span\u003e\u003cspan address=\"10.1186/1475-2875-8-239\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHape EE, Ngonyani AT, Mabula DM, Nkya JD, Thomas CA, Omari MJ, et al. Delayed mating in the malaria vector Anopheles funestus compared to Anopheles arabiensis (Diptera: Culicidae). J Med Entomol. 2025;62(4):921\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jme/tjaf059\u003c/span\u003e\u003cspan address=\"10.1093/jme/tjaf059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Malaria vector, Mosquito rearing, Sub-Saharan Africa, Residual malaria, egg production","lastPublishedDoi":"10.21203/rs.3.rs-9291181/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9291181/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAnopheles funestus\u003c/em\u003e s.s plays a major role in sustaining residual malaria transmission in many African settings. Despite its epidemiological importance, research on its biology and developing effective control strategies is greatly hindered by the persistent challenges associated with the establishing of stable colonies in laboratory. This study capitalises a successful process for establishing \u003cem\u003eAn. funestus\u003c/em\u003e strain in laboratory population in Burkina Faso (FUNBF).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAdult female mosquitoes were collected over a two-year period in Di\u0026eacute;bougou and Soumousso using mouth and battery-powered electric aspirators. These mosquitoes were morphologically identified as belonging to the \u003cem\u003eAn. funestus\u003c/em\u003e group. Individual females were allowed to oviposit separately in 200mL disposable cups. Female mosquitoes were then confirmed at the molecular level as \u003cem\u003eAn. funestus\u003c/em\u003e s.s. Progeny were reared under standard laboratory conditions. Fecundity and egg hatch rate were recorded for founder (F0) females.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 1,871 female mosquitoes were collected across two years to establish the colony. F0 mean fecundity was 85\u0026plusmn; 2.9 eggs per female (range: 35\u0026ndash;124) with a mean hatching rate of 88.6% (range: 66.4\u0026ndash;100%). FUNBF has been maintained successfully for 23 generations with sustained egg production.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis work represents a significant advance in the domestication of \u003cem\u003eAn. funestus\u003c/em\u003e, one of the main malaria vectors in sub-Saharan Africa.\u003c/p\u003e","manuscriptTitle":"From the field to the laboratory: establishment of Anopheles funestus s.s. colony in Burkina Faso","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-10 19:32:45","doi":"10.21203/rs.3.rs-9291181/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T17:14:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T16:28:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T12:30:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245976935979883350848857740323890553723","date":"2026-04-09T08:20:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288344769569749350696529643008149994557","date":"2026-04-08T21:14:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311238243493681574605436523576599677640","date":"2026-04-08T07:34:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265017923630511885660468125683191959136","date":"2026-04-07T08:18:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190287019537306928528548867640937878785","date":"2026-04-06T16:04:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-05T14:59:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T13:04:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-03T12:54:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2026-04-01T11:19:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd54a878-b02c-45d6-bc44-b9e4fad16210","owner":[],"postedDate":"April 10th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-29T17:14:36+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T17:24:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-10 19:32:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9291181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9291181","identity":"rs-9291181","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}