Visual cues enhance mating success in laboratory colonies of the malaria vector Anopheles funestus with strain-specific responses

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Mrosso, Ashley M. Burke, Halfan S. Ngowo, Megan A. Riddin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6527720/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Establishing and maintaining laboratory colonies of the malaria vector, Anopheles funestus colonies, using wild-collected material, has proven challenging, in part because of their low propensity to mate in captivity. This study assessed how cage conditions influence the mating success of two Anopheles funestus strains, originally sourced from different geographic areas, Angola (FANG) and Mozambique (FUMOZ). Methods The visual environment in adult mosquito-rearing cages was manipulated either by covering the cages with black cloth to create artificial horizons or by placing contrasting black swarming markers at various positions inside the cages. Mating success was assessed by dissecting the spermathecal capsules of the females after they were reared for 10 days in the cages. Results Overall, mating success was higher in the FANG compared to FUMOZ females, both under artificial horizons (OR = 2.2, 95% CI: [1.83, 2.64]) and visual swarming markers (OR = 3.37, 95% CI: [2.53, 4.49]). Covering the mosquito cages with black opaque cloth and placing a contrasting marker inside the cage increased mating success for both FANG (χ 2 = 27.6, p < 0.001), and FUMOZ (χ 2 = 27.6, p < 0.001) compared to the standard uncovered cage. However, the two An. funestus strains responded differently to the same adult holding conditions. In the FUMOZ strain, mating success increased when the top half of the cage was covered with black cloth (OR = 1.70, 95% CI: 1.18–2.46) or when a contrasting marker was placed at the cage base (OR = 1.98, 95% CI: 1.38–2.85). In contrast, the FANG strain showed improved mating success when the cage side was covered (OR = 2.06, 95% CI: 1.40–3.02). Conclusion This study demonstrates that manipulating the visual environment within adult mosquito-rearing cages can significantly enhance mating success in An. funestus , though the effectiveness of specific visual cues varies between strains. While both FANG and FUMOZ responded positively to visual enhancements, their differing responses to the same conditions underscore the importance of tailoring rearing protocols to the geographic origin of the strain. These findings offer practical guidance for improving the colonization and maintenance of An. funestus in laboratory settings, which is critical for advancing research on this major malaria vector. Anopheles funestus Anopheles insectary artificial horizons cage size mating success rates rearing cage swarming markers Figures Figure 1 Figure 2 Figure 3 BACKGROUND Anopheles funestus (a member of the An. funestus group) plays a major role in malaria transmission in Africa [ 1 – 7 ]. There have been several attempts to establish sustained laboratory colonies of this species to support studies of its biology and control, but success has been elusive [ 8 , 9 , 5 , 10 ]. Previously, only two Anopheles funestus strains, one from Angola (FANG) and another from Mozambique (FUMOZ), had been successfully colonized in the laboratory from wild-caught populations [ 9 , 11 ]. However, more recently, a strain from Tanzania was also colonized [ 10 ]. The FANG and FUMOZ strains have been widely used as models in studies to understand (amongst other studies) the physiology and life history of An. funestus to enhance and optimise the rearing challenges under insectary conditions [ 9 , 5 , 10 – 14 ]. However, the low mating success in captivity has been identified to be the main hindrance towards establishing and sustaining An. funestus under laboratory conditions [ 5 , 10 , 7 ]. Like other Anopheles mosquitoes, An. funestus females generally mate with males in swarms, which are influenced by specific environmental features that shape local light conditions [ 15 – 17 ]. Considering that Anopheles mosquito swarming activities occur during sunset and at a specific location in nature [ 15 , 18 – 20 , 16 , 17 ], various natural and artificial environmental features can alter local light conditions in ways that trigger swarming (Fig. 1 ). There are also surface or ground markers such as soil colour and the presence or absence of objects that have been associated with An. funestus swarms. To enhance the establishment and sustainability of malaria vectors in captivity, several environmental conditions such as temperature and relative humidity have been studied and adopted in rearing facilities [ 8 , 9 , 22 , 23 ]. Artificial lighting systems that mimic the natural photo periodism of 12:12 hours light:dark cycle including a dusk and dawn period have been used in Anopheles insectaries [ 8 , 9 , 22 ], but these conditions have not significantly improved the colonization of An. funestus group species compared to those in the An. gambiae complex [ 8 , 5 ]. Swarm induction has also been achieved both with simulated artificial horizons in laboratory cages in An. gambiae and An. arabiensis [ 24 , 25 ], and light contrasting markers, dimming lights and other natural swarm markers in large semi-field experiment cages/chambers [ 26 , 27 , 19 , 28 ]. Cage covering with a black cloth may have a noticeable effect on the light environment inside rearing cages and has so far been demonstrated to improve the longevity and mating of reared An. arabiensis [ 25 ]. However, to the best of our knowledge, there is no established standard mosquito-rearing cage for the successful mating of An. funestus. Establishing conditions that will improve the mating success of An. funestus in rearing cages might prove a major tool in enhancing its colonisation attempts. In this study, we report on the impact of manipulating the environment inside the adult rearing cages by the inclusion of artificial light horizons and light contrasting visual markers on the mating success of two An. funestus strains originated from different geographical locations. MATERIALS AND METHODS Experimental site Experiments were conducted in the Botha de Meillon insectary (BDMI) located at the National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. The insectary climate is automated at 26 ± 2°C temperature, 70 ± 10% relative humidity, and a 12:12 hours light: dark (or day: night) phase cycle. A set of three 42-watt halogen bulbs (Pick n Pay brand, South Africa) are located on the ceiling and illuminate gradually over a 30-minute period to simulate the onset of sunrise and sunset (dimming) and remain lit during the day/light phase. Eight light-emitting diode (LED) tube lights (F58W/GRO-T8, Germany) are located underneath the rearing shelves to illuminate the larval rearing bowls (but not the adult cages) to enable working (feeding larvae and harvesting adults) in the larval stages in the insectary. These lights switch on 30 minutes after sunrise and 30 min before sunset is initiated by the ceiling lights. Anopheles funestus used in the study The two colonised strains of An. funestus used in this study were established in the early 2000s from field-collected mosquitoes from two geographical locations; one from Mozambique (denoted as FUMOZ) and the other from Angola (denoted as FANG) [ 5 ]. Even though both the FANG and FUMOZ strains belong to the MW molecular lineage of An. funestus , they differ at a finer population structure level [ 29 , 5 ]. That is, FANG belongs only to clade 1, while FUMOZ includes individuals from both clade 1 and clade II [ 29 , 5 ]. Adult FUMOZ strains were obtained from a colony that was routinely reared in square BugDorm cages (30 × 30 × 30 cm) (MegaView Science Co. Ltd., Taichung, Taiwan). Adult FANG strains were obtained from a colony that was routinely reared in 20-litre non-transparent white cylindrical polypropylene bucket cages with a clear fine mesh top cover and a round side hole covered by a clear fine-meshed sleeve for transferring material into and out of the cage. Both FUMOZ and FANG adult mosquitoes were reared in densities of approximately 1,500 to 2,000 mosquitoes per cage and held on a 10% sugar solution that was changed twice a week. Visual cues and cage alterations The effect of horizon lighting was assessed by comparing the mating success of An. funestus females placed into five experimental BugDorm cages (15 × 15 x 15 cm, height x length x width) (MegaView Science Co. Ltd., Taichung, Taiwan) that were covered with a black opaque cloth to create different planes of horizons: one cage was fully covered, two cages were half covered in horizontal to simulate top and bottom horizon, one cage was half covered in vertical plane to simulate vertical horizon, and one cage was completely uncovered to serve as a control (Fig. 2 ). Similarly, the effect of artificial visual markers was evaluated by using four experimental BugDorm cages (15 cm × 15 cm × 15 cm) (MegaView Science Co. Ltd., Taichung, Taiwan). The visual markers were simulated using a round black paper card (7 cm diameter) that was fixed to either the centre of the base, left hand side, or top side of three separate cages respectively, and the fourth cage was without a visual marker to serve as a control (Fig. 2 ). Experiment setting Male and female pupae (Fig. 3 A and B) were sorted under an Olympus dissecting microscope (OLYMPUS SZX7, model SZ2-ILST, Tokyo, Japan), and placed in a plastic bowl (12 cm, diameter and 5 cm, height) to emerge. A total of 100 An. funestus FANG or FUMOZ pupae at 1:1 male: female ratio were sorted and placed in separate plastic bowls (12 cm, 5 cm; diameter, height). The bowls were put in separate experimental cages with access to 10% sugar solution for the emergent mosquitoes. The emerged adult mosquitoes were allowed 10 days, to mate, with a change of sugar water every two days [ 30 ]. Six biological replicates of the experiment were conducted using different mosquito generational cohorts and only one cage per biological replicate was set for each treatment. At the end of the experiment (day 10) surviving females were removed from the cages and their spermathecal capsule dissected. Spermathecal dissection Spermatheca dissections were done as described in the Methods in Anopheles Research (MR4 [ 31 ], with some modifications where mosquitoes were first knocked down in a paper cup covered with a net in a -20°C freezer for 10 minutes. Mosquitoes were mounted on a clean microscope slide with a drop of distilled water and the last abdominal segment was removed using a pair of fine forceps under an Olympus stereo microscope (OLYMPUS SZX7, model SZ2-ILST, Tokyo, Japan) to isolate the spermatheca capsule. The mosquito corpses were discarded and the spermatheca capsule was observed under an Olympus light microscope (OLYMPUS BX50, model BX50F4, Tokyo, Japan) with 40X magnification on the Stream Essentials 1.9.4 Olympus soft image solutions. Spermatheca capsules from females with grey content were recorded as “mated” while a clear spermatheca was scored as “not mated” (Fig. 3 C and D). Data analysis The mean insemination values for both FUMOZ and FANG strains were estimated using a generalised linear mixed model in RStudio (version 4.2.2) with the lme4 package (version 1.1–32) [ 32 ]. Mating successes were modelled as binomial variable with cage modification as a fixed categorical independent variable, experimental replicate as the random effect, and the standard cage was set as a reference group. The final model was constructed with the following function; Mating success ~ Cage specifications + (1|EXP_Replicate)-1, family = binomial (link = "logit"), data = data ). Log likelihood ratio tests (LRT) were used to test the significance of each variable of interest in all models. RESULTS The effect of artificial horizons on the mating success of An. funestus Covering adult An. funestus rearing cages with a black opaque cloth to create artificial horizons significantly influenced the mating status of FUMOZ (χ 2 = 27.6, p < 0.001), and FANG (χ 2 = 27.6, p < 0.001). When compared to the uncovered control cage, the FUMOZ females had significantly higher mating success when housed in the top-covered cage (48.3%, n = 230, p < 0.01, Table 1 ). The FANG strain had significantly higher mating success in the side-covered (79.2%, n = 278, p < 0.001, Table 1 ) and top-covered cages (72.6%, n = 272, p < 0.05, Table 1 ) compared to the control cage (Table 1 ). The fully covered cage had significantly lower insemination ratio of 25.6% (n = 229) for FUMOZ, but even though it had the lowest insemination ratio 61.0% (n = 279) for FANG, it was significant when compared to the uncovered cage (Table 1 ). Generally, the FANG strain had higher mating success rates than the FUMOZ strain across all categories of artificial horizon experiments, OR = 2.2, 95% CI: [1.83, 2.64]). Table 1 Mating success rate (% insemination) of An. funestus FUMOZ and FANG strains housed in adult rearing cages modified to create different artificial horizon planes. Strain Cage Modification Sample Size (N) % Insemination [95% CI] OR [95% CI] P -Values FUMOZ No Cover 252 35.4 [28.7, 42.6] 1 Full Cover 229 25.6 [19.7, 32.5] 0.63 [0.42, 0.93] 0.021 Bottom Cover 240 37.1 [30.2, 44.6] 1.08 [0.74, 1.56] 0.689 Side Cover 238 31.7 [25.2, 38.9] 0.85 [0.58, 1.24] 0.387 Top Cover 230 48.3 [40.7, 55.9] 1.70 [1.18, 2.46] 0.004 FANG No Cover 275 64.9 [56.1, 72.8] 1 Full Cover 273 61.0 [52.0, 69.3] 0.85 [0.60, 1.20] 0.350 Bottom Cover 253 67.2 [58.3, 74.9] 1.11 [0.77, 1.59] 0.587 Side Cover 278 79.2 [71.9, 85.0] 2.06 [1.40, 3.02] < 0.001 Top Cover 272 72.6 [64.6, 79.8] 1.45 [1.01, 2.10] 0.046 CI – Confidence interval, OR – Odd ratios, bold font indicates statistically significant difference from the control cage. The impact of a dark contrast visual marker on the mating success of An. funestus The inclusion of artificial dark-contrast visual markers significantly influenced the mating success rate of FUMOZ (χ 2 = 18.7, p < 0.001), but not FANG (χ 2 = 1.96, p = 0.58). The FUMOZ strain housed in the cage with a visual marker placed on the bottom base had significantly higher mating success ratios (49.5%, n = 262, p < 0.001, Table 2 ) compared to the control cage with no marker. Generally, the odds mating success were higher for FANG strain compared to FUMOZ strain in all categories of light contrast markers (OR = 3.37, 95% CI: [2.53, 4.49], Table 2 ). Table 2 Mating success ratios (% insemination) of An. funestus FUMOZ and FANG strains held in adult rearing cages with black light-contrast visual markers positioned on different sides of the cage. Strain Visual Marker Sample Size % Insemination [95% CI] OR [95% CI] P- Values FUMOZ No Marker 243 33.1 [26.5, 40.5] 1 Side Marker 268 34.8 [28.3, 42.2] 1.08 [0.75, 1.56] 0.682 Top Marker 256 34.9 [28.3, 42.2] 1.08 [0.74, 1.58] 0.670 Bottom Marker 262 49.5 [42.2, 56.9] 1.98 [1.38, 2.85] < 0.001 FANG No Marker 265 79.9 [71.3, 86.4] 1 Side Marker 247 75.3 [65.8, 82.9] 0.77 [0.51, 1.17] 0.220 Top Marker 258 76.2 [66.9, 83.6] 0.81 [0.53, 1.23] 0.317 Bottom Marker 260 78.8 [69.9, 85.6] 0.93 [0.61, 1.43] 0.752 CI – Confidence interval, OR – Odd ratios, bold font indicates statistically significant difference from the control cage. DISCUSSION Attempts to optimize conditions for colonisation and maintenance of An. funestus colonies in captivity are being carried out in different locations [ 33 , 5 , 10 ]. Maximizing the mating success rates of An. funestus will complement the optimisations done in larvae rearing [ 14 ] and adult feeding [ 12 ] that have proved to improve the physiological fitness of the vector in captivity. The findings from this study provide insight into the important modifications that can improve the mating rates of An. funestus in insectary rearing cages. Moreover, the two strains of An. funestus used in this study expressed varied responses in terms of mating success rates under similar cage modifications. The top covering of the cage to create a bottom horizon favoured mating of the FUMOZ strain while the side covering favoured the FANG strain mating success rate. Likewise, the inclusion of visual markers in adult An. funestus cage significantly improved the mating success of the FUMOZ strain but did not have any notable impact on the mating of the FANG strain compared to the controls. These modifications are a vital guide for new attempts to colonise this significant malaria vector. The observed improved mating success rates might suggest increased swarming activities induced by cage covering and the light contrasting visual marker. This observation corresponds with field observations of the association of Anopheles mosquito swarms with the presence of varied ground markers including surfaces such as open-ground areas [ 34 , 15 , 21 , 20 , 16 , 17 ], burnt ground, and garbage heaps [ 20 ]. Based on these observations, it is suggested that swarming, and hence mating behaviours, are influenced by the presence of surfaces that may contrast the fading light at sunset. Furthermore, the presences of environmental features such as vegetation [ 15 , 16 ], termite mounds [ 20 ], and man-made structures such as houses [ 34 , 15 , 17 ] have been associated with An. funestus swarming. These features and other landforms, such as hills, affect the natural horizon at sunset. Therefore, covering the top or vertical half of the mosquito cages in the insectary might have simulated these light horizons inside the cage that induced mating. Considering the natural preference by An. funestus to rest in dark places in their habitats [ 8 , 35 , 22 ], the top and side cage covering may have created a dark resting area in relation to the ceiling lights of the insectary room, allowing mosquitoes to rest and reserve additional energy required for increased mating activities. Covering mosquito cages has been shown to improve mating and survival time in An. arabiensis [ 25 ], which might be due to the dark space created by the cover that is conducive to resting and hence, increases energy reserves [ 36 ]. Limited light environment inside the fully covered cage might have interrupted the mosquito circadian clock leading to the lowest mating success compared to the rest of the cage modifications. Several studies have reported the importance of changing light phases on the mosquito circadian-clock-dependent behaviour which include mating [ 37 – 40 ]. The fully covered cage would have been expected to create a dark space that would ensure mosquitoes had conditions conducive for resting. However, the observed low mating success in the fully covered cages demonstrates the importance light and changes in the light environment have on the mating activity of An. funestus . The inclusion of artificial visual markers in the form of black discs significantly increased the mating success of An. funestus FUMOZ strain. A visual marker placed on the base of the cage might have induced mating activities by contrasting the white base of the cage. The effect of the base marker on the FUMOZ strain corresponds with field observations with ground markers [ 15 , 18 , 21 , 41 ]. Like the observed effect of base visual markers on the FUMOZ strain mating success, light contrasting surfaces have been demonstrated to induce swarming activities in An. gambiae mosquitoes under semi-field settings [ 27 , 28 ]. The FANG strain which originated in Angola (insecticide susceptible) had consistently higher mating rates compared to the FUMOZ strain (metabolic pyrethroid resistant strain) originated from Mozambique [ 9 ]. The highest mating success rates of the FANG strain in this study were about 80% which is slightly higher compared to the previous mean mating rates of 74.8% reported on the same species that were also maintained in a cylindrical cage [ 11 ]. However, this study observed 100 mosquitoes in BugDorm cages (15 x 15 x 15 cm, height x length x width) (equivalent to three litres) compared to the 600 mosquitoes in a five-litre cage in the previous study by Zengenene [ 11 ]. Different optimisations on Anopheles mating in captivity have found cage size and mosquito densities to be an important factor in improving mating [ 8 , 42 , 24 , 31 ]. The highest observed mating success rates for the FUMOZ strain when the experimental cages were not modified (control cages) in this study were lower (35%) than reported from previous studies (more than 70%) on the same species [ 10 , 12 , 30 ]. However, in the two previous studies [ 10 , 12 ], large cubic cages (30 x 30 cm) were used, and a randomly selected subsample of 10 females from the experimental cage were dissected for insemination, while for this study, mating rates were evaluated in small cubic cages (15 x 15 cm) and all surviving females from the 50 female per cage used were dissected after the allowed mating period. Even though Maharaj [ 30 ] used the experimental BugDorm cages (15 x 15 cm) as in this study, they started their experiments with newly emerged mosquitoes allowing them to mate for 10 to 12 days. Experiments in this study were set with mosquitoes at the pupae stage and mating was evaluated after 10 days. Pupae were used to ensure adult mosquitoes would emerge and stay in specific experimental conditions of cage modification and mosquito density. The observed mating success differences between the two strains might be due to their varied geographical originality or other evolutionary genomic differences. Several studies have reported evidence of population segregation influenced by geographical landmarks and ground distance between the two populations [ 43 – 45 , 7 ]. Similarly, field observations have reported notable differences in swarming behaviour within the same species of Anopheles mosquitoes across different geographical regions [ 21 , 46 , 47 ]. For example, Anopheles gambiae swarms in the south-eastern side of the African continent [ 21 , 47 ], were observed to differ in swarm timing, height from the ground and swarm markers from those on the western side of Africa [ 21 , 48 ]. Moreover, it has been observed that mosquito populations of the same species from different geographical conditions or localities may express fine genomic differences that can influence their adaptation and survival, especially against insecticides [ 49 , 7 ]. These genomic differences have been reported to affect the physiological and reproductive fitness of Anopheles mosquitoes. For example, the presence of target-site mutations for insecticide resistance has been linked to lowering the reproductive fitness of male mosquitoes [ 50 ]. The Anopheles funestus FANG strain is known to be susceptible to insecticides while FUMOZ expresses metabolic resistance [ 9 , 5 , 51 – 55 ]. Expression of metabolic resistance has been linked to reducing the reproductive fitness in An. funestus FUMOZ strain [ 52 , 53 ]. Therefore, the observed difference in the two strains of the same species might be connected to their difference in geographical origin or other underlying genomic differences including their susceptibility to insecticides. However, it is unlikely that the observed decrease in insemination rates for the FUMOZ strain was due to susceptibility status since Zengenene [ 11 ] and Felamboahangy [ 12 ] reported higher mating success rates using the same strains in the BDMI. During this study, South Africa experienced major electrical supply shortages, which impacted the climatic control systems in the BDMI. It is possible that the two strains are affected differently due to these environmental stressors. This highlights the importance of future studies to investigate the impact of environmental changes on this species as it might have implications for climate change. Furthermore, the main FUMOZ colony at BDMI is maintained in 20-litre non-transparent white cylindrical polypropylene bucket cages. However, for this study a sub colony of FUMOZ was established and maintained in BugDorm cages (30 × 30 × 30 cm) (MegaView Science Co. Ltd., Taichung, Taiwan), while the FANG stain was obtained from the main colony which is maintained in 20-litre non-transparent white cylindrical polypropylene bucket cages. Therefore, the electrical supply challenges and the differences in colony maintenance cages might have affected the strains’ acclimatisation to the lab conditions hence impacting their response to the manipulation of experimental cages. Conclusion This study highlights the critical role of light and visual cues in influencing the mating success of An. funestus in laboratory settings. Manipulating the light environment within rearing cages, through artificial horizons and visual markers, significantly improved mating outcomes, underscoring the importance of cage design in facilitating successful copulation. Notably, the two strains tested, FUMOZ and FANG, responded differently to the same visual modifications. These differences likely reflect underlying genetic or physiological variation linked to their distinct geographical origins. As such, standardised rearing conditions may not be universally effective across all An. funestus populations. To improve colony establishment and maintenance, especially when working with wild-derived strains or planning mass-rearing for release programmes, it is essential to tailor adult holding conditions to the specific characteristics of each strain. Customizing the visual and environmental features of insectary cages can help accommodate subtle inter-strain differences that impact reproductive fitness. These findings have practical implications for vector research and control initiatives, particularly in the context of producing large numbers of mosquitoes for experimental or operational use. They also underscore the broader need to account for intra-species variation when designing protocols for colonising malaria vectors in captivity. Abbreviations BDMI: Botha de Meillon insectary; FANG: Anopheles funestus s.s. from Angola; FUMOZ: Anopheles funestus s.s. from Mozambique; NICD: National Institute for Communicable Diseases; VCRL: Vector Control Reference Declarations Availability of data and materials The dataset for this study is available from the corresponding author upon request. Ethics approval Ethical approval for this project was obtained from the University of the Witwatersrand Animal Ethics Research Committee ethics (Certificate reference 20190701-70). Competing interests The authors declare that they have no competing interests. Funding This work was made possible through financial support under the Bill & Melinda Gates Foundation Grant (OPP1177156) awarded to the Ifakara Health Institute and Partners including the University of the Witwatersrand, National Research Foundation of South Africa to LLK (SRUG2203311457) and the Jennifer Ward Oppenheimer Research Grant (02) awarded to BC. Authors contributions LLK conceived the study, PCM, AMB, LLK, and BWTC were involved in designing this study. PCM performed data collection. PCM and HN conducted data analysis. PCM wrote the manuscript draft. LLK, BWTC, MR, AMB, HN and FOO provided a thorough review of the manuscript. 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Light manipulation of mosquito behaviour: acute and sustained photic suppression of biting activity in the Anopheles gambiae malaria mosquito. Parasites & Vectors. 2017;10(1). Marchand R. A new cage for observing mating behavior of wild Anopheles gambiae in the laboratory. J Am Mosq Control Assoc. 1985;1(2):234–236. Mashatola T, Munhenga G, Koekemoer LL. Evaluating different cages for rearing Anopheles arabiensis (Diptera: Culicidae) under laboratory conditions. African Entomology. 2017;25(2):534–539. Paton D, Toure M, Sacko A, Coulibaly MB, Traore SF, Tripet F. Genetic and environmental factors associated with laboratory rearing affect survival and assortative mating but not overall mating success in Anopheles gambiae sensu stricto. PLoS ONE. 2013;8(12):e82631. Facchinelli L, Valerio L, Lees RS, Oliva CF, Persampieri T, Collins CM, et al. Stimulating Anopheles gambiae swarms in the laboratory: application for behavioural and fitness studies. Malaria Journal. 2015;14(1):271. Niang A, Nignan C, Serge Poda B, Sawadogo SP, Roch Dabiré K, Gnankiné O, et al. Semi-field and indoor setups to study malaria mosquito swarming behavior. Parasites & Vectors. 2019;12(1):1–9. Choi KS, Coetzee M, Koekemoer LL. Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay). Parasites & Vectors. 2013;6:173. Maharaj S, Ekoka E, Erlank E, Nardini L, Reader J, Birkholtz L-M, et al. The ecdysone receptor regulates several key physiological factors in Anopheles funestus. Malaria Journal. 2022;21(1):97. MR4. Methods in Anopheles Research Atlanta, GA. 2015 [2015 Edition:[Available from: https://www.beiresources.org/portals/2/MR4/MR4_Publications/Methods%20in%20Anopheles%20Research% 202014/2014MethodsinAnophelesResearchManualFullVersionv2tso.pdf . Bates D, Mächler M, Bolker B, Walker S. Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software. 2015;67(1):1–48. Service MW, Oguamah D. Colonization of Anopheles funestus . Nature. 1958;181(4617):1225–1225. Harper J. Notes on the Swarming of Males of Anopheles. funestus (Giles), in East Africa. East African Medical Journal. 1944;21(5). Clements AN. The biology of mosquitoes. United Kingdoms: CABI Publishing Wallingford; 1999. 752 p. Takken W, Charlwood D, Lindsay SW. The behaviour of adult Anopheles gambiae, sub-Saharan Africa’s principal malaria vector, and its relevance to malaria control: a review. Malaria Journal. 2024;23(1):161. Jones MDR, Hill M, Hope AM. The circadian flight activity of the mosquito, Anopheles gambiae : Phase Setting by the Light Regime. Journal of Experimental Biology. 1967;47(3):503–511. Baik LS, Nave C, Au DD, Guda T, Chevez JA, Ray A, et al. Circadian regulation of light-evoked attraction and avoidance behaviors in daytime- versus nighttime-biting mosquitoes. Current Biology. 2020;30(16):3252–3259 e3253. Manoukis NC. Drivers of mosquito mating: Light cycle genes and environmental and chemical cues modulate male mosquito swarming. Science. 2021;371(6527):340–341. Liu X, Yang S, Yao Y, Wu S, Wu P, Zhai Z. Opsin1 regulates light-evoked avoidance behavior in Aedes albopictus . BMC Biology. 2022;20(1). Diabaté A, Yaro AS, Dao A, Diallo M, Huestis DL, Lehmann T. Spatial distribution and male mating success of Anopheles gambiae swarms. BMC Evolutionary Biology. 2011;11(1):184. Pan J, Yu T, Zhu H. Study on laboratory breeding of Anopheles balabacensis balabacensis Baisas. 1982. Koekemoer LL, Kamau L, Garros C, Manguin S, Hunt RH, Coetzee M. Impact of the Rift Valley on restriction fragment length polymorphism typing of the major African malaria vector Anopheles funestus (Diptera: Culicidae). Journal of Medical Entomology. 2006;43(6):1178–1184. Amvongo-Adjia N, Riveron JM, Njiokou F, Wanji S, Wondji CS. Influence of a major mountainous landscape barrier (Mount Cameroon) on the spread of metabolic ( GSTe2 ) and Target-Site ( Rdl ) resistance alleles in the African malaria vector Anopheles funestus . Genes (Basel). 2020;11(12). Kaddumukasa MA, Wright J, Muleba M, Stevenson JC, Norris DE, Coetzee M. Genetic differentiation and population structure of Anopheles funestus from Uganda and the southern African countries of Malawi, Mozambique, Zambia and Zimbabwe. Parasit & Vectors. 2020;13(1):87. Sawadogo SP, Costantini C, Pennetier C, Diabaté A, Gibson G, Dabiré RK. Differences in timing of mating swarms in sympatric populations of Anopheles coluzzii and Anopheles gambiae s.s. (formerly An. gambiae M and S molecular forms) in Burkina Faso, West Africa. Parasites & Vectors. 2013;6(1):275. Baeshen R. Swarming Behavior in Anopheles gambiae (sensu lato): Current Knowledge and Future Outlook Journal of Medical Entomology. 2022;59(1):56–66. Gueye OK, Niang A, Faye MB, Dia AK, Ahmed AA, Sy O, et al. Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal. Journal of Medical Entomology. 2023;60(6):1278–1287. Miles AJ. Genomic epidemiology of malaria vectors in the Anopheles gambiae species complex: University of Oxford; 2021. Platt N, Kwiatkowska RM, Irving H, Diabaté A, Dabire R, Wondji CS. Target-site resistance mutations ( kdr and RDL ), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae . Heredity. 2015;115(3):243–252. Venter N, Oliver SV, Muleba M, Davies C, Hunt RH, Koekemoer LL, et al. Benchmarking insecticide resistance intensity bioassays for Anopheles malaria vector species against resistance phenotypes of known epidemiological significance. Parasites & Vectors. 2017;10(1):198. Tchouakui M, Riveron JM, Djonabaye D, Tchapga W, Irving H, Takam PS, et al. Fitness Costs of the Glutathione S-Transferase Epsilon 2 ( L119F-GSTe2 ) Mediated Metabolic Resistance to Insecticides in the Major African Malaria Vector Anopheles funestus . Genes (Basel). 2018;9(12):645. Tchouakui M, Mugenzi LMJ, Wondji MJ, Tchoupo M, Njiokou F, Wondji CS. Combined over-expression of two cytochrome P450 genes exacerbates the fitness cost of pyrethroid resistance in the major African malaria vector Anopheles funestus . Pestic Biochem Physiol. 2021;173:104772. Menze BD, Mugenzi LMJ, Tchouakui M, Wondji MJ, Tchoupo M, Wondji CS. Experimental hut trials reveal that CYP6P9a /b P450 Alleles are reducing the efficacy of pyrethroid-only olyset net against the malaria vector Anopheles funestus but PBO-based olyset plus net remains effective. Pathogens. 2022;11(6):638. Nolden M, Paine MJI, Nauen R. Sequential phase I metabolism of pyrethroids by duplicated CYP6P9 variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito Anopheles funestus . Insect Biochem Mol Biol. 2022;148:103813. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-6527720","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454355036,"identity":"dd3462ed-0a69-4b87-9222-f1e9e487d5ce","order_by":0,"name":"Paul C. Mrosso","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYBADOTYJxgbStBjDtEgQrSWxAaqWsBZzsbMPH/74Y5PeJ93cwPBxD0OdPCH3Wc5ONzbmbUvLbZM52MA44xkDYS8Z3E5jk2ZsOJzbJpHYwMxzgEGCmZDDQFokf/w5nM4G08JGjBYJHrbDCXAtPIS0WM5OYwb5xRDksIMzDkhIziCkxVw6jREUYvLyM9IfPvhwwIafYIgZIHMOEBWTBoSVjIJRMApGwYgHALh6NjUQtuqzAAAAAElFTkSuQmCC","orcid":"","institution":"University of the Witwatersrand","correspondingAuthor":true,"prefix":"","firstName":"Paul","middleName":"C.","lastName":"Mrosso","suffix":""},{"id":454355037,"identity":"e85c6648-9b9b-4826-923a-e6decf8ac4bf","order_by":1,"name":"Ashley M. Burke","email":"","orcid":"","institution":"University of the Witwatersrand","correspondingAuthor":false,"prefix":"","firstName":"Ashley","middleName":"M.","lastName":"Burke","suffix":""},{"id":454355038,"identity":"4f51ba1f-9a3f-4b55-9fd3-0062f754bb9b","order_by":2,"name":"Halfan S. Ngowo","email":"","orcid":"","institution":"Ifakara Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Halfan","middleName":"S.","lastName":"Ngowo","suffix":""},{"id":454355039,"identity":"4a65fa90-1920-44a1-a666-276170048a1b","order_by":3,"name":"Megan A. Riddin","email":"","orcid":"","institution":"University of Pretoria","correspondingAuthor":false,"prefix":"","firstName":"Megan","middleName":"A.","lastName":"Riddin","suffix":""},{"id":454355040,"identity":"4e2a3eae-25f9-40f6-9250-93b7c1439b98","order_by":4,"name":"Fredros O. Okumu","email":"","orcid":"","institution":"Ifakara Health Institute","correspondingAuthor":false,"prefix":"","firstName":"Fredros","middleName":"O.","lastName":"Okumu","suffix":""},{"id":454355041,"identity":"e7c94d47-2034-43ee-8189-96ca7dc8d170","order_by":5,"name":"Bernard W.T. Coetzee","email":"","orcid":"","institution":"University of Pretoria","correspondingAuthor":false,"prefix":"","firstName":"Bernard","middleName":"W.T.","lastName":"Coetzee","suffix":""},{"id":454355042,"identity":"3760aa29-ba7f-4adc-8802-dfe0a4f698d7","order_by":6,"name":"Lizette L. Koekemoer","email":"","orcid":"","institution":"University of the Witwatersrand","correspondingAuthor":false,"prefix":"","firstName":"Lizette","middleName":"L.","lastName":"Koekemoer","suffix":""}],"badges":[],"createdAt":"2025-04-25 09:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6527720/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6527720/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82507276,"identity":"a807c15e-f752-4cab-b1a0-422add7b6df9","added_by":"auto","created_at":"2025-05-12 09:53:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105420,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic illustration of different features such as \u003cstrong\u003e1)\u003c/strong\u003e trees, \u003cstrong\u003e2)\u003c/strong\u003e mountains, \u003cstrong\u003e3)\u003c/strong\u003e rivers, and \u003cstrong\u003e4)\u003c/strong\u003ebuilt structures such as houses that may impact the properties of sun light dusk in a way that creates hypothetical spots with a favourable light environment for Anopheles mosquito swarms (red circles denoted ‘S’). The diagram is a stereotypical illustration of natural scenarios based on various narratives in the literature created by considering environmental features that may contrast or form horizons against rays of light at sunset.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6527720/v1/5ffdfb7fa31462e326393712.jpg"},{"id":82507308,"identity":"521d776f-2673-462f-9395-558cdb8aee7c","added_by":"auto","created_at":"2025-05-12 09:53:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":422863,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of adult mosquito rearing cage with standard sleeve opening and black cloth coverings: \u003cstrong\u003eA)\u003c/strong\u003e fully covered, \u003cstrong\u003eB)\u003c/strong\u003e half covered horizontal top horizon, \u003cstrong\u003eC)\u003c/strong\u003e half covered horizontal bottom horizon, \u003cstrong\u003eD)\u003c/strong\u003ehalf covered vertical horizon, and \u003cstrong\u003eE)\u003c/strong\u003e not covered (control cage), \u003cstrong\u003eF)\u003c/strong\u003e swarm marker fixed on the base of the cage, \u003cstrong\u003eG)\u003c/strong\u003eswarm marker fixed on one side of the cage, \u003cstrong\u003eH)\u003c/strong\u003e swarm marker fixed on the top of the cage, and \u003cstrong\u003eI)\u003c/strong\u003e no swarm marker (control cage).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6527720/v1/be909b591c778f9183ccc7cf.jpg"},{"id":82507261,"identity":"7b2c2973-ed9d-4189-a144-05a077cf4493","added_by":"auto","created_at":"2025-05-12 09:53:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1031328,"visible":true,"origin":"","legend":"\u003cp\u003eAnopheles pupae and spermatheca after dissection as seen under an Olympus dissecting microscope and light microscope respectively, \u003cstrong\u003eA)\u003c/strong\u003e Male pupae with a sharp claw at the end of the last abdominal segment, \u003cstrong\u003eB)\u003c/strong\u003eFemale pupae with no sharp claw at the tip of the last abdominal segment\u003cstrong\u003e C)\u003c/strong\u003eAn inseminated spermatheca with the grey matter (sperm cells) inside, \u003cstrong\u003eD)\u003c/strong\u003ean un-inseminated spermatheca.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6527720/v1/050f08b7f78bea502294ee23.jpg"},{"id":89508397,"identity":"f92e5121-9f96-405d-84b7-a7ab092f93d5","added_by":"auto","created_at":"2025-08-20 17:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2500681,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6527720/v1/65c3ecda-4fed-4120-a313-17d14df1f8d2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visual cues enhance mating success in laboratory colonies of the malaria vector Anopheles funestus with strain-specific responses","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003e \u003cem\u003eAnopheles funestus\u003c/em\u003e (a member of the \u003cem\u003eAn. funestus\u003c/em\u003e group) plays a major role in malaria transmission in Africa [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. There have been several attempts to establish sustained laboratory colonies of this species to support studies of its biology and control, but success has been elusive [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previously, only two \u003cem\u003eAnopheles funestus\u003c/em\u003e strains, one from Angola (FANG) and another from Mozambique (FUMOZ), had been successfully colonized in the laboratory from wild-caught populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, more recently, a strain from Tanzania was also colonized [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The FANG and FUMOZ strains have been widely used as models in studies to understand (amongst other studies) the physiology and life history of \u003cem\u003eAn. funestus\u003c/em\u003e to enhance and optimise the rearing challenges under insectary conditions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the low mating success in captivity has been identified to be the main hindrance towards establishing and sustaining \u003cem\u003eAn. funestus\u003c/em\u003e under laboratory conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLike other \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes, \u003cem\u003eAn. funestus\u003c/em\u003e females generally mate with males in swarms, which are influenced by specific environmental features that shape local light conditions [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Considering that \u003cem\u003eAnopheles\u003c/em\u003e mosquito swarming activities occur during sunset and at a specific location in nature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], various natural and artificial environmental features can alter local light conditions in ways that trigger swarming (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There are also surface or ground markers such as soil colour and the presence or absence of objects that have been associated with \u003cem\u003eAn. funestus\u003c/em\u003e swarms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo enhance the establishment and sustainability of malaria vectors in captivity, several environmental conditions such as temperature and relative humidity have been studied and adopted in rearing facilities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Artificial lighting systems that mimic the natural photo periodism of 12:12 hours light:dark cycle including a dusk and dawn period have been used in \u003cem\u003eAnopheles\u003c/em\u003e insectaries [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but these conditions have not significantly improved the colonization of \u003cem\u003eAn. funestus\u003c/em\u003e group species compared to those in the \u003cem\u003eAn. gambiae\u003c/em\u003e complex [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Swarm induction has also been achieved both with simulated artificial horizons in laboratory cages in \u003cem\u003eAn. gambiae\u003c/em\u003e and \u003cem\u003eAn. arabiensis\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and light contrasting markers, dimming lights and other natural swarm markers in large semi-field experiment cages/chambers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cage covering with a black cloth may have a noticeable effect on the light environment inside rearing cages and has so far been demonstrated to improve the longevity and mating of reared \u003cem\u003eAn. arabiensis\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, to the best of our knowledge, there is no established standard mosquito-rearing cage for the successful mating of \u003cem\u003eAn. funestus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eEstablishing conditions that will improve the mating success of \u003cem\u003eAn. funestus\u003c/em\u003e in rearing cages might prove a major tool in enhancing its colonisation attempts. In this study, we report on the impact of manipulating the environment inside the adult rearing cages by the inclusion of artificial light horizons and light contrasting visual markers on the mating success of two \u003cem\u003eAn. funestus\u003c/em\u003e strains originated from different geographical locations.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental site\u003c/h2\u003e \u003cp\u003eExperiments were conducted in the Botha de Meillon insectary (BDMI) located at the National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. The insectary climate is automated at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C temperature, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, and a 12:12 hours light: dark (or day: night) phase cycle. A set of three 42-watt halogen bulbs (Pick n Pay brand, South Africa) are located on the ceiling and illuminate gradually over a 30-minute period to simulate the onset of sunrise and sunset (dimming) and remain lit during the day/light phase. Eight light-emitting diode (LED) tube lights (F58W/GRO-T8, Germany) are located underneath the rearing shelves to illuminate the larval rearing bowls (but not the adult cages) to enable working (feeding larvae and harvesting adults) in the larval stages in the insectary. These lights switch on 30 minutes after sunrise and 30 min before sunset is initiated by the ceiling lights.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnopheles funestus used in the study\u003c/h3\u003e\n\u003cp\u003eThe two colonised strains of \u003cem\u003eAn. funestus\u003c/em\u003e used in this study were established in the early 2000s from field-collected mosquitoes from two geographical locations; one from Mozambique (denoted as FUMOZ) and the other from Angola (denoted as FANG) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Even though both the FANG and FUMOZ strains belong to the MW molecular lineage of \u003cem\u003eAn. funestus\u003c/em\u003e, they differ at a finer population structure level [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. That is, FANG belongs only to clade 1, while FUMOZ includes individuals from both clade 1 and clade II [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Adult FUMOZ strains were obtained from a colony that was routinely reared in square BugDorm cages (30 \u0026times; 30 \u0026times; 30 cm) (MegaView Science Co. Ltd., Taichung, Taiwan). Adult FANG strains were obtained from a colony that was routinely reared in 20-litre non-transparent white cylindrical polypropylene bucket cages with a clear fine mesh top cover and a round side hole covered by a clear fine-meshed sleeve for transferring material into and out of the cage. Both FUMOZ and FANG adult mosquitoes were reared in densities of approximately 1,500 to 2,000 mosquitoes per cage and held on a 10% sugar solution that was changed twice a week.\u003c/p\u003e\n\u003ch3\u003eVisual cues and cage alterations\u003c/h3\u003e\n\u003cp\u003eThe effect of horizon lighting was assessed by comparing the mating success of \u003cem\u003eAn. funestus\u003c/em\u003e females placed into five experimental BugDorm cages (15 \u0026times; 15 x 15 cm, height x length x width) (MegaView Science Co. Ltd., Taichung, Taiwan) that were covered with a black opaque cloth to create different planes of horizons: one cage was fully covered, two cages were half covered in horizontal to simulate top and bottom horizon, one cage was half covered in vertical plane to simulate vertical horizon, and one cage was completely uncovered to serve as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, the effect of artificial visual markers was evaluated by using four experimental BugDorm cages (15 cm \u0026times; 15 cm \u0026times; 15 cm) (MegaView Science Co. Ltd., Taichung, Taiwan). The visual markers were simulated using a round black paper card (7 cm diameter) that was fixed to either the centre of the base, left hand side, or top side of three separate cages respectively, and the fourth cage was without a visual marker to serve as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExperiment setting\u003c/h3\u003e\n\u003cp\u003eMale and female pupae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B) were sorted under an Olympus dissecting microscope (OLYMPUS SZX7, model SZ2-ILST, Tokyo, Japan), and placed in a plastic bowl (12 cm, diameter and 5 cm, height) to emerge. A total of 100 \u003cem\u003eAn. funestus\u003c/em\u003e FANG or FUMOZ pupae at 1:1 male: female ratio were sorted and placed in separate plastic bowls (12 cm, 5 cm; diameter, height). The bowls were put in separate experimental cages with access to 10% sugar solution for the emergent mosquitoes. The emerged adult mosquitoes were allowed 10 days, to mate, with a change of sugar water every two days [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Six biological replicates of the experiment were conducted using different mosquito generational cohorts and only one cage per biological replicate was set for each treatment. At the end of the experiment (day 10) surviving females were removed from the cages and their spermathecal capsule dissected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSpermathecal dissection\u003c/h3\u003e\n\u003cp\u003eSpermatheca dissections were done as described in the Methods in \u003cem\u003eAnopheles\u003c/em\u003e Research (MR4 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], with some modifications where mosquitoes were first knocked down in a paper cup covered with a net in a -20\u0026deg;C freezer for 10 minutes. Mosquitoes were mounted on a clean microscope slide with a drop of distilled water and the last abdominal segment was removed using a pair of fine forceps under an Olympus stereo microscope (OLYMPUS SZX7, model SZ2-ILST, Tokyo, Japan) to isolate the spermatheca capsule. The mosquito corpses were discarded and the spermatheca capsule was observed under an Olympus light microscope (OLYMPUS BX50, model BX50F4, Tokyo, Japan) with 40X magnification on the Stream Essentials 1.9.4 Olympus soft image solutions. Spermatheca capsules from females with grey content were recorded as \u0026ldquo;mated\u0026rdquo; while a clear spermatheca was scored as \u0026ldquo;not mated\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe mean insemination values for both FUMOZ and FANG strains were estimated using a generalised linear mixed model in \u003cem\u003eRStudio\u003c/em\u003e (version 4.2.2) with the \u003cem\u003elme4\u003c/em\u003e package (version 1.1\u0026ndash;32) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Mating successes were modelled as binomial variable with cage modification as a fixed categorical independent variable, experimental replicate as the random effect, and the standard cage was set as a reference group. The final model was constructed with the following function; Mating success\u0026thinsp;~\u0026thinsp;Cage specifications + (1|EXP_Replicate)-1, family\u0026thinsp;=\u0026thinsp;binomial (link = \"logit\"), data\u0026thinsp;=\u0026thinsp;data ). Log likelihood ratio tests (LRT) were used to test the significance of each variable of interest in all models.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of artificial horizons on the mating success of An. funestus\u003c/h2\u003e \u003cp\u003eCovering adult \u003cem\u003eAn. funestus\u003c/em\u003e rearing cages with a black opaque cloth to create artificial horizons significantly influenced the mating status of FUMOZ (χ\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;27.6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and FANG (χ\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;27.6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When compared to the uncovered control cage, the FUMOZ females had significantly higher mating success when housed in the top-covered cage (48.3%, n\u0026thinsp;=\u0026thinsp;230, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The FANG strain had significantly higher mating success in the side-covered (79.2%, n\u0026thinsp;=\u0026thinsp;278, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and top-covered cages (72.6%, n\u0026thinsp;=\u0026thinsp;272, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) compared to the control cage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The fully covered cage had significantly lower insemination ratio of 25.6% (n\u0026thinsp;=\u0026thinsp;229) for FUMOZ, but even though it had the lowest insemination ratio 61.0% (n\u0026thinsp;=\u0026thinsp;279) for FANG, it was significant when compared to the uncovered cage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Generally, the FANG strain had higher mating success rates than the FUMOZ strain across all categories of artificial horizon experiments, OR\u0026thinsp;=\u0026thinsp;2.2, 95% CI: [1.83, 2.64]).\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\u003eMating success rate (% insemination) of \u003cem\u003eAn. funestus\u003c/em\u003e FUMOZ and FANG strains housed in adult rearing cages modified to create different artificial horizon planes.\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=\"left\" 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=\"left\" 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\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCage Modification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSample Size (N)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Insemination [95% CI]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOR [95% CI]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-Values\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eFUMOZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.4 [28.7, 42.6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFull Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.6 [19.7, 32.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.63 [0.42, 0.93]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBottom Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.1 [30.2, 44.6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.08 [0.74, 1.56]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.689\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSide Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.7 [25.2, 38.9]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.85 [0.58, 1.24]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.387\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTop Cover\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e230\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e48.3 [40.7, 55.9]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.70 [1.18, 2.46]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eFANG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64.9 [56.1, 72.8]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFull Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.0 [52.0, 69.3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.85 [0.60, 1.20]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBottom Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.2 [58.3, 74.9]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.11 [0.77, 1.59]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.587\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSide Cover\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e278\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e79.2 [71.9, 85.0]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e2.06 [1.40, 3.02]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTop Cover\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e272\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e72.6 [64.6, 79.8]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.45 [1.01, 2.10]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.046\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eCI \u0026ndash;\u003c/b\u003e Confidence interval, \u003cb\u003eOR \u0026ndash;\u003c/b\u003e Odd ratios, bold font indicates statistically significant difference from the control cage.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of a dark contrast visual marker on the mating success of An. funestus\u003c/h2\u003e \u003cp\u003eThe inclusion of artificial dark-contrast visual markers significantly influenced the mating success rate of FUMOZ (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;18.7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not FANG (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.96, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.58). The FUMOZ strain housed in the cage with a visual marker placed on the bottom base had significantly higher mating success ratios (49.5%, n\u0026thinsp;=\u0026thinsp;262, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) compared to the control cage with no marker. Generally, the odds mating success were higher for FANG strain compared to FUMOZ strain in all categories of light contrast markers (OR\u0026thinsp;=\u0026thinsp;3.37, 95% CI: [2.53, 4.49], 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\u003eMating success ratios (% insemination) of \u003cem\u003eAn. funestus\u003c/em\u003e FUMOZ and FANG strains held in adult rearing cages with black light-contrast visual markers positioned on different sides of the cage.\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=\"left\" 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=\"left\" 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\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVisual Marker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSample Size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Insemination [95% CI]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOR [95% CI]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP-\u003c/em\u003eValues\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFUMOZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.1 [26.5, 40.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSide Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.8 [28.3, 42.2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.08 [0.75, 1.56]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTop Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.9 [28.3, 42.2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.08 [0.74, 1.58]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.670\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBottom Marker\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e262\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e49.5 [42.2, 56.9]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.98 [1.38, 2.85]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFANG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e79.9 [71.3, 86.4]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSide Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.3 [65.8, 82.9]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.77 [0.51, 1.17]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.220\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTop Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76.2 [66.9, 83.6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.81 [0.53, 1.23]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.317\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBottom Marker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78.8 [69.9, 85.6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.93 [0.61, 1.43]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.752\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eCI \u0026ndash;\u003c/b\u003e Confidence interval, \u003cb\u003eOR \u0026ndash;\u003c/b\u003e Odd ratios, bold font indicates statistically significant difference from the control cage.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAttempts to optimize conditions for colonisation and maintenance of \u003cem\u003eAn. funestus\u003c/em\u003e colonies in captivity are being carried out in different locations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Maximizing the mating success rates of \u003cem\u003eAn. funestus\u003c/em\u003e will complement the optimisations done in larvae rearing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and adult feeding [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] that have proved to improve the physiological fitness of the vector in captivity. The findings from this study provide insight into the important modifications that can improve the mating rates of \u003cem\u003eAn. funestus\u003c/em\u003e in insectary rearing cages. Moreover, the two strains of \u003cem\u003eAn. funestus\u003c/em\u003e used in this study expressed varied responses in terms of mating success rates under similar cage modifications. The top covering of the cage to create a bottom horizon favoured mating of the FUMOZ strain while the side covering favoured the FANG strain mating success rate. Likewise, the inclusion of visual markers in adult \u003cem\u003eAn. funestus\u003c/em\u003e cage significantly improved the mating success of the FUMOZ strain but did not have any notable impact on the mating of the FANG strain compared to the controls. These modifications are a vital guide for new attempts to colonise this significant malaria vector.\u003c/p\u003e \u003cp\u003eThe observed improved mating success rates might suggest increased swarming activities induced by cage covering and the light contrasting visual marker. This observation corresponds with field observations of the association of \u003cem\u003eAnopheles\u003c/em\u003e mosquito swarms with the presence of varied ground markers including surfaces such as open-ground areas [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], burnt ground, and garbage heaps [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on these observations, it is suggested that swarming, and hence mating behaviours, are influenced by the presence of surfaces that may contrast the fading light at sunset. Furthermore, the presences of environmental features such as vegetation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], termite mounds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and man-made structures such as houses [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] have been associated with \u003cem\u003eAn. funestus\u003c/em\u003e swarming. These features and other landforms, such as hills, affect the natural horizon at sunset. Therefore, covering the top or vertical half of the mosquito cages in the insectary might have simulated these light horizons inside the cage that induced mating.\u003c/p\u003e \u003cp\u003eConsidering the natural preference by \u003cem\u003eAn. funestus\u003c/em\u003e to rest in dark places in their habitats [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the top and side cage covering may have created a dark resting area in relation to the ceiling lights of the insectary room, allowing mosquitoes to rest and reserve additional energy required for increased mating activities. Covering mosquito cages has been shown to improve mating and survival time in \u003cem\u003eAn. arabiensis\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which might be due to the dark space created by the cover that is conducive to resting and hence, increases energy reserves [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Limited light environment inside the fully covered cage might have interrupted the mosquito circadian clock leading to the lowest mating success compared to the rest of the cage modifications. Several studies have reported the importance of changing light phases on the mosquito circadian-clock-dependent behaviour which include mating [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The fully covered cage would have been expected to create a dark space that would ensure mosquitoes had conditions conducive for resting. However, the observed low mating success in the fully covered cages demonstrates the importance light and changes in the light environment have on the mating activity of \u003cem\u003eAn. funestus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe inclusion of artificial visual markers in the form of black discs significantly increased the mating success of \u003cem\u003eAn. funestus\u003c/em\u003e FUMOZ strain. A visual marker placed on the base of the cage might have induced mating activities by contrasting the white base of the cage. The effect of the base marker on the FUMOZ strain corresponds with field observations with ground markers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Like the observed effect of base visual markers on the FUMOZ strain mating success, light contrasting surfaces have been demonstrated to induce swarming activities in \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes under semi-field settings [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe FANG strain which originated in Angola (insecticide susceptible) had consistently higher mating rates compared to the FUMOZ strain (metabolic pyrethroid resistant strain) originated from Mozambique [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The highest mating success rates of the FANG strain in this study were about 80% which is slightly higher compared to the previous mean mating rates of 74.8% reported on the same species that were also maintained in a cylindrical cage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, this study observed 100 mosquitoes in BugDorm cages (15 x 15 x 15 cm, height x length x width) (equivalent to three litres) compared to the 600 mosquitoes in a five-litre cage in the previous study by Zengenene [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Different optimisations on \u003cem\u003eAnopheles\u003c/em\u003e mating in captivity have found cage size and mosquito densities to be an important factor in improving mating [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe highest observed mating success rates for the FUMOZ strain when the experimental cages were not modified (control cages) in this study were lower (35%) than reported from previous studies (more than 70%) on the same species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, in the two previous studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], large cubic cages (30 x 30 cm) were used, and a randomly selected subsample of 10 females from the experimental cage were dissected for insemination, while for this study, mating rates were evaluated in small cubic cages (15 x 15 cm) and all surviving females from the 50 female per cage used were dissected after the allowed mating period. Even though Maharaj [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] used the experimental BugDorm cages (15 x 15 cm) as in this study, they started their experiments with newly emerged mosquitoes allowing them to mate for 10 to 12 days. Experiments in this study were set with mosquitoes at the pupae stage and mating was evaluated after 10 days. Pupae were used to ensure adult mosquitoes would emerge and stay in specific experimental conditions of cage modification and mosquito density.\u003c/p\u003e \u003cp\u003eThe observed mating success differences between the two strains might be due to their varied geographical originality or other evolutionary genomic differences. Several studies have reported evidence of population segregation influenced by geographical landmarks and ground distance between the two populations [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, field observations have reported notable differences in swarming behaviour within the same species of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes across different geographical regions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For example, \u003cem\u003eAnopheles gambiae\u003c/em\u003e swarms in the south-eastern side of the African continent [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], were observed to differ in swarm timing, height from the ground and swarm markers from those on the western side of Africa [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, it has been observed that mosquito populations of the same species from different geographical conditions or localities may express fine genomic differences that can influence their adaptation and survival, especially against insecticides [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These genomic differences have been reported to affect the physiological and reproductive fitness of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes. For example, the presence of target-site mutations for insecticide resistance has been linked to lowering the reproductive fitness of male mosquitoes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The \u003cem\u003eAnopheles funestus\u003c/em\u003e FANG strain is known to be susceptible to insecticides while FUMOZ expresses metabolic resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Expression of metabolic resistance has been linked to reducing the reproductive fitness in \u003cem\u003eAn. funestus\u003c/em\u003e FUMOZ strain [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, the observed difference in the two strains of the same species might be connected to their difference in geographical origin or other underlying genomic differences including their susceptibility to insecticides. However, it is unlikely that the observed decrease in insemination rates for the FUMOZ strain was due to susceptibility status since Zengenene [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Felamboahangy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] reported higher mating success rates using the same strains in the BDMI.\u003c/p\u003e \u003cp\u003eDuring this study, South Africa experienced major electrical supply shortages, which impacted the climatic control systems in the BDMI. It is possible that the two strains are affected differently due to these environmental stressors. This highlights the importance of future studies to investigate the impact of environmental changes on this species as it might have implications for climate change. Furthermore, the main FUMOZ colony at BDMI is maintained in 20-litre non-transparent white cylindrical polypropylene bucket cages. However, for this study a sub colony of FUMOZ was established and maintained in BugDorm cages (30 \u0026times; 30 \u0026times; 30 cm) (MegaView Science Co. Ltd., Taichung, Taiwan), while the FANG stain was obtained from the main colony which is maintained in 20-litre non-transparent white cylindrical polypropylene bucket cages. Therefore, the electrical supply challenges and the differences in colony maintenance cages might have affected the strains\u0026rsquo; acclimatisation to the lab conditions hence impacting their response to the manipulation of experimental cages.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the critical role of light and visual cues in influencing the mating success of \u003cem\u003eAn. funestus\u003c/em\u003e in laboratory settings. Manipulating the light environment within rearing cages, through artificial horizons and visual markers, significantly improved mating outcomes, underscoring the importance of cage design in facilitating successful copulation. Notably, the two strains tested, FUMOZ and FANG, responded differently to the same visual modifications. These differences likely reflect underlying genetic or physiological variation linked to their distinct geographical origins. As such, standardised rearing conditions may not be universally effective across all \u003cem\u003eAn. funestus\u003c/em\u003e populations. To improve colony establishment and maintenance, especially when working with wild-derived strains or planning mass-rearing for release programmes, it is essential to tailor adult holding conditions to the specific characteristics of each strain. Customizing the visual and environmental features of insectary cages can help accommodate subtle inter-strain differences that impact reproductive fitness. These findings have practical implications for vector research and control initiatives, particularly in the context of producing large numbers of mosquitoes for experimental or operational use. They also underscore the broader need to account for intra-species variation when designing protocols for colonising malaria vectors in captivity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBDMI: Botha de Meillon insectary; FANG: \u003cem\u003eAnopheles funestus s.s.\u003c/em\u003e from Angola; FUMOZ: \u003cem\u003eAnopheles funestus s.s.\u003c/em\u003e from Mozambique; NICD: National Institute for Communicable Diseases; VCRL: Vector Control Reference\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset for this study is available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this project was obtained from the University of the Witwatersrand Animal Ethics Research Committee ethics (Certificate reference 20190701-70).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was made possible through financial support under the Bill \u0026amp; Melinda Gates Foundation Grant (OPP1177156) awarded to the Ifakara Health Institute and Partners including the University of the Witwatersrand, National Research Foundation of South Africa to LLK (SRUG2203311457) and the Jennifer Ward Oppenheimer Research Grant (02) awarded to BC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLLK conceived the study, PCM, AMB, LLK, and BWTC were involved in designing this study. PCM performed data collection. PCM and HN conducted data analysis. PCM wrote the manuscript draft. LLK, BWTC, MR, AMB, HN and FOO provided a thorough review of the manuscript. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors information (optional)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorresponding author\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLizette Leonie Koekemoer: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also thank Prince Zengenene from the Vector Control Reference (VCRL) Laboratory at the National Institute for Communicable Diseases for assisting with FANG pupae, Mr Zilindile Zulu, Christine Moletsane, and Duma Mnisi for assisting with blood feeding of adult mosquitoes, and Prof Basil Brooke and VCRL staff for hosting and providing support during the insectary work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoss R. 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Parasites \u0026amp; Vectors. 2017;10(1):198.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTchouakui M, Riveron JM, Djonabaye D, Tchapga W, Irving H, Takam PS, \u003cem\u003eet al.\u003c/em\u003e Fitness Costs of the Glutathione S-Transferase Epsilon 2 (\u003cem\u003eL119F-GSTe2\u003c/em\u003e) Mediated Metabolic Resistance to Insecticides in the Major African Malaria Vector \u003cem\u003eAnopheles funestus\u003c/em\u003e. Genes (Basel). 2018;9(12):645.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTchouakui M, Mugenzi LMJ, Wondji MJ, Tchoupo M, Njiokou F, Wondji CS. Combined over-expression of two cytochrome \u003cem\u003eP450\u003c/em\u003e genes exacerbates the fitness cost of pyrethroid resistance in the major African malaria vector \u003cem\u003eAnopheles funestus\u003c/em\u003e. Pestic Biochem Physiol. 2021;173:104772.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenze BD, Mugenzi LMJ, Tchouakui M, Wondji MJ, Tchoupo M, Wondji CS. Experimental hut trials reveal that \u003cem\u003eCYP6P9a\u003c/em\u003e/b P450 Alleles are reducing the efficacy of pyrethroid-only olyset net against the malaria vector \u003cem\u003eAnopheles funestus\u003c/em\u003e but PBO-based olyset plus net remains effective. Pathogens. 2022;11(6):638.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNolden M, Paine MJI, Nauen R. Sequential phase I metabolism of pyrethroids by duplicated \u003cem\u003eCYP6P9\u003c/em\u003e variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito \u003cem\u003eAnopheles funestus\u003c/em\u003e. Insect Biochem Mol Biol. 2022;148:103813.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anopheles funestus, Anopheles insectary, artificial horizons, cage size, mating success rates, rearing cage, swarming markers","lastPublishedDoi":"10.21203/rs.3.rs-6527720/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6527720/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEstablishing and maintaining laboratory colonies of the malaria vector, \u003cem\u003eAnopheles funestus\u003c/em\u003e colonies, using wild-collected material, has proven challenging, in part because of their low propensity to mate in captivity. This study assessed how cage conditions influence the mating success of two \u003cem\u003eAnopheles funestus\u003c/em\u003e strains, originally sourced from different geographic areas, Angola (FANG) and Mozambique (FUMOZ).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe visual environment in adult mosquito-rearing cages was manipulated either by covering the cages with black cloth to create artificial horizons or by placing contrasting black swarming markers at various positions inside the cages. Mating success was assessed by dissecting the spermathecal capsules of the females after they were reared for 10 days in the cages.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOverall, mating success was higher in the FANG compared to FUMOZ females, both under artificial horizons (OR\u0026thinsp;=\u0026thinsp;2.2, 95% CI: [1.83, 2.64]) and visual swarming markers (OR\u0026thinsp;=\u0026thinsp;3.37, 95% CI: [2.53, 4.49]). Covering the mosquito cages with black opaque cloth and placing a contrasting marker inside the cage increased mating success for both FANG (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;27.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and FUMOZ (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;27.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the standard uncovered cage. However, the two \u003cem\u003eAn. funestus\u003c/em\u003e strains responded differently to the same adult holding conditions. In the FUMOZ strain, mating success increased when the top half of the cage was covered with black cloth (OR\u0026thinsp;=\u0026thinsp;1.70, 95% CI: 1.18\u0026ndash;2.46) or when a contrasting marker was placed at the cage base (OR\u0026thinsp;=\u0026thinsp;1.98, 95% CI: 1.38\u0026ndash;2.85). In contrast, the FANG strain showed improved mating success when the cage side was covered (OR\u0026thinsp;=\u0026thinsp;2.06, 95% CI: 1.40\u0026ndash;3.02).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study demonstrates that manipulating the visual environment within adult mosquito-rearing cages can significantly enhance mating success in \u003cem\u003eAn. funestus\u003c/em\u003e, though the effectiveness of specific visual cues varies between strains. While both FANG and FUMOZ responded positively to visual enhancements, their differing responses to the same conditions underscore the importance of tailoring rearing protocols to the geographic origin of the strain. These findings offer practical guidance for improving the colonization and maintenance of \u003cem\u003eAn. funestus\u003c/em\u003e in laboratory settings, which is critical for advancing research on this major malaria vector.\u003c/p\u003e","manuscriptTitle":"Visual cues enhance mating success in laboratory colonies of the malaria vector Anopheles funestus with strain-specific responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 09:53:16","doi":"10.21203/rs.3.rs-6527720/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62b33b4d-b13b-4e05-8c3c-acaec4b6759a","owner":[],"postedDate":"May 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-20T17:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-12 09:53:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6527720","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6527720","identity":"rs-6527720","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}