Distance from human dwellings differentially affects the efficacy of a synthetic cattle urine odour lure to trap malaria vectors
Malaria Journal volume 22, Article number: 8 (2023)
Cost–effective outdoor–based devices for surveillance and control of outdoor mosquito vector populations can substantially improve their efficacy when baited with synthetic human and animal odours. This study aimed at assessing the dose–dependent efficacy of a previously developed synthetic cattle urine odour to lure malaria vectors, and other mosquito species, to traps placed at different distances from human dwellings outdoors.
The efficacy of the cattle urine odour lure was assessed through a 5 × 5 Latin square design, using two sets of 5 Suna traps placed at either 1.5 m or 5 m from an adjacent human dwelling, in the rural village of Sagamaganga, Tanzania. Each trap was deployed with one of four doses of the synthetic cattle urine odour blend or a solvent control (heptane). Traps were rotated daily so that each dose and control visited each position twice over a period of 20 experimental nights. The relative attractiveness of each treatment dose and control was compared using a generalized linear mixed model for each species caught.
A total of 1568 mosquitoes were caught, of which 783 were anophelines and 785 were culicines. Of the anophelines, 41.6 and 58.3% were primary and secondary vector species, respectively. Unfed and fed females of the primary vector, Anopheles arabiensis, were caught dose–dependently, close to human dwellings (1.5 m), whereas unfed, fed and gravid secondary vector Anopheles pharoensis females were caught dose–dependently, but at a farther distance from the dwellings (5 m). Females of Culex spp. were caught dose–dependently in similar numbers irrespective of the distance from human dwellings.
This study further clarifies the factors to be considered for the implementation of outdoor trapping using the synthetic cattle urine lure to target exophilic and exophagic malaria vectors, for which efficient surveillance and control tools are currently lacking. The findings resulting from this study make significant progress in providing the needed information to overcome the regulatory obstacles to make this tool available for integrated vector management programs, including registration, as well as evaluation and regulation by the World Health Organization.
Outdoor–based devices are required to complement current surveillance and frontline control tools [1,2,3], as a result of changes in biting times, and in the composition of outdoor–biting malaria vector species. These factors increasingly contribute to persistent malaria transmission in sub–Saharan Africa and beyond [2, 4,5,6]. Cost–effective outdoor–based devices for surveillance and control of outdoor mosquito vector populations can substantially improve their efficacy, when baited with synthetic human and animal odours [7,8,9,10]. While several synthetic blends mimicking human odour (e.g., Mbita, BG, Ifakara lures) have been developed, these are limited in their deployment, as these only lure host-seeking females, and require the addition of carbon dioxide, which is difficult to procure and prohibitively expensive in remote areas [11,12,13]. Thus, alternative odour-based lures targeting males and females at other physiological states need to be assessed for their efficacy.
Cattle urine attracts many haematophagous insects [14, 15], with fresh and aged cattle urine demonstrated to attract various physiological stages and species of mosquitoes [16,17,18]. Adult female malaria vectors use the urine as a supplementary nitrogen–rich meal, enhancing flight mobility, survival and reproductive traits . The drive for locating nitrogen-rich resources by malaria vectors can be harnessed for vector and malaria control, as shown by , in which a synthetic cattle urine odour blend was used to lure host seeking, blood fed and gravid females into traps in a rural, malaria endemic region in southern Ethiopia. Further proof concerning the attractiveness of the synthetic cattle urine odour to malaria vectors in different settings with various trap placements relative to human dwellings is, however, needed to confirm and improve its utility as a surveillance and control tool in integrated vector management.
In this study, the efficacy of the synthetic cattle urine odour lure in attracting malaria vectors outdoors was evaluated in the Kilombero Valley, Tanzania, by placing traps close to and away from human dwellings. The perspective of using the synthetic cattle urine odour blend in improving surveillance and control of outdoor biting malaria vectors is discussed.
The study was conducted in Sagamaganga village (S 8° 3′ 50.352″ E 36° 47′ 46.254″), Kilombero Valley, south–eastern Tanzania at an altitude of 300 m above sea level (Fig. 1). The average annual temperature ranges between 20 °C and 32 °C, with an annual rainfall between 1200 mm and 1800 mm [21, 22]. The area experiences two main seasons, the wet and dry, which extend from February to June and July to January, respectively. The main economic activities are agricultural, including rice cultivation and livestock keeping. The common domesticated animals are predominantly cattle, goats, sheep, chickens and dogs, with cattle as the most abundant. Anopheles arabiensis and Anopheles coustani have been identified as the most abundant primary and secondary vectors of malaria, respectively . The malaria prevalence rate in Kilombero valley has decreased from 14% in 2007–2011 [22, 24] to 0.4% in 2019 (Kyeba et al., pers. commun.).
Selection and characteristics of study households
Ten study households were selected randomly from a register of all houses by using global positioning system coordinates of 152 households in the study village in simple random sampling (Fig. 1) . All study households had mud–brick walls and open eaves. Three households had thatched roofs, while the remaining seven were made of corrugated iron. All selected study households had at least one cattle shed located approximate 50 m to 100 m from the house (Fig. 1). The number of occupants per household varied from one to five. Each household was provided with one-to-three new long-lasting insecticidal nets (Olyset, A to Z Textiles Mills, Arusha, Tanzania) depending on the number of beds. In all the study households, no animals were kept inside the human dwellings, and cooking was exclusively conducted outside.
Assessing the effect of dose and trapping distance of the synthetic cattle urine odour
The attractiveness of the synthetic cattle urine odour lure  against wild mosquitoes was assessed through a repeated 5 × 5 Latin square design, using Suna traps® (Biogents AG, Germany). Two sets of five traps, placed at either 1.5 m or 5 m from the household, were evaluated during each experimental night. The traps were suspended approximately 20 cm above the ground, 50–100 m away from any adjacent cattle shed, and run overnight from 18h00 to 06h00. Each trap was deployed with the synthetic cattle urine odour blend: 7:9:156:156:1:4; 2-cyclohexen-1-one (96% GC grade VWR, Stockholm, Sweden): phenol: p–cresol: m–cresol: decanal: linalool (> 95.0% GC grade, Sigma Aldrich Sweden AB, Stockholm)  at one of four doses (0.1, 0.03, 0.01, 0.003 volume/ volume) or a solvent control (heptane, 97.0% solvent GC grade, Sigma Aldrich). The odour blend and heptane (2 ml) were dispensed via wick dispensers, which allow the release of all compounds in constant ratios throughout the night . Initially, the treatments and controls were randomly assigned to the ten houses, then subsequently rotated nightly in serial order, so that each dose and control visited each trapping position twice over a period of 20 experimental nights. This trial was conducted in four rounds: 5 nights in April, 5 nights in May, and 10 nights in June 2021.
Adult mosquito identification
Caught anopheline mosquitoes were preserved in 1.5 ml Eppendorf tubes containing silica gel while awaiting further laboratory analyses. The mosquitoes were identified using morphological identification keys [27, 28], and sorted based on sex and physiological state (abdominal status: unfed, fed, semi-gravid and gravid). Molecular species identification, using multiplexed polymerase chain reaction, was performed on all collected An. gambiae sensu lato (s.l.) (n = 232) [28, 30] and Anopheles funestus s.l. (n = 23) [29, 31, 32].
Daily variation in mosquito captures across different doses of the synthetic odour blend were analysed using generalized linear mixed models with R statistical software version 3.6.2 . Since the data were zero–inflated and over–dispersed, as confirmed by the Shapiro test, a negative binomial distribution was employed . “Dose”, “Trap distance” and “Household IDs” were treated as fixed effects, while sampling night was treated as a random effect. Separate analyses were performed for each mosquito species.
Mosquitoes caught and identified
A total of 1568 mosquitoes were caught and identified, of which 783 (648 females, 135 males) were anophelines and 785 (577 females, 208 males) were culicines. Among the anophelines, 41.6% were primary malaria vector species, while 58.3% were secondary vector species (Table 1). The primary malaria vectors collected were An. arabiensis (92.9%; n = 560) and An. funestus sensu stricto (s.s.) (7.1%; n = 39). The three main secondary malaria vector species collected were An. pharoensis (72.7%; n = 618), An. coustani (12.9%; n = 113), and Anopheles tenebrosus (11.0%; n = 46) (Table 1). As mirrored in the total collections, the physiological status of most female anophelines caught were unfed (551) followed by blood fed (54), and gravid mosquitoes (11) (Table 1). Among the culicines, 99.0% (n = 777) were Culex species while 1.0% (n = 8) were Mansonia and Aedes species (Table 1).
Dose and distance from dwellings affect trap captures of malaria vectors
The attractiveness of the synthetic cattle urine odour lure to both the primary and secondary malaria vector species varied with dose and trapping distance from the human dwelling (Table 1). For all of the primary malaria vectors caught, irrespective of physiological state, the lure attracted a significantly higher proportion of An. arabiensis (n = 229) relative to An. funestus s.s. (n = 16), irrespective of the dose and trapping distance (χ12 = 86.512, P < 0.001). Anopheles arabiensis were caught more frequently next to the human dwellings (1.5 m) in a dose–dependent manner (GLMM: P = 0.001) compared to those further away (5 m; GLMM: P < 0.001) (Fig. 2A). While the number of An. funestus s.s. caught were too few to assess statistically, this species appeared to be caught more frequently at the higher doses tested, irrespective of distance from the human dwelling (Table 1). Of the secondary malaria vectors (Table 1), only An. pharoensis were caught in sufficient numbers for statistical analysis. This species was caught more frequently at a trapping distance of 5 m from the dwellings (GLMM: P = 0.044) in a dose-dependent manner (GLMM: P = 0.001) (Fig. 2B). Anopheles pharoensis appears to be more sensitive to the lure, as a higher number of individuals were caught at a lower dose than An. arabiensis (Fig. 2).
Dose and distance from dwellings affect trap captures of Culex species
The attractiveness of the synthetic cattle urine odour lure to Culex species varied with dose, irrespective of trapping distance from the human dwellings (Table 1). Culex spp. were caught in a dose–dependent manner with more individuals caught in traps baited with the two highest doses (GLMM: P = 0.011 and P = 0.036, respectively).
The deployment of cattle urine–based attractants has the potential to improve outdoor mosquito surveillance and control tools [17,18,19,20]. In this study, the importance of trap placement in achieving the optimal efficacy of the cattle urine lure for targeting various species of both primary and secondary malaria vectors, as well as other vector and nuisance species, e.g., Culex spp., is demonstrated. The findings, furthermore, demonstrate a species– and dose–dependent response towards the cattle urine lure. The results of this study are discussed in relation to further challenges to be addressed before this lure may be implemented in future integrated vector management programs.
The trapping distance from human dwellings significantly affected the number and species of mosquitoes caught in traps baited with the synthetic cattle urine lure. Females of the primary vector, An. arabiensis, both unfed and fed, were predominantly caught closer to the dwellings, while the majority of females of the secondary vector, An. pharoensis, unfed, fed and gravid, were caught at the farthest distance from the dwellings tested. This trend in the species profile caught close to the dwellings has been observed previously, which may have led researchers to conclude, erroneously, that cattle urine odour is not very attractive to An. pharoensis females [17,18,19,20]. In contrast to the anophelines, Culex spp. were caught in similar numbers irrespective of the distance from human dwellings. This finding emphasizes that the fine–scale spatial heterogeneity in the landscape is an important species-dependent driver of movement patterns of mosquitoes, as previously demonstrated for both anophelines and culicines [34, 35]. Thus, the efficacy of outdoor odour–baited traps relies on their proper deployment in the landscape, and a detailed understanding of the ecology of the mosquito species targeted.
Anopheles arabiensis, An. pharoensis and Culex spp. appeared to differ in their sensitivity to the synthetic cattle urine lure, with An. pharoensis likely being the most sensitive, and Culex spp. appeared to be attracted to the highest doses of the lure. Both Anopheles species were caught in lower numbers in traps that were baited with the highest dose of the lure, demonstrating a similar behavioural response as that of tsetse flies to various doses of cattle urine odour . The rationale for the observed species–specific variation in behavioural response is likely due to differences in perception of the bioactive volatile organic compounds in the synthetic cattle urine odour. Whether adaptive selection has affected the association between select mosquito species and cattle urine remains to be explored.
This study provides additional essential information on the synthetic cattle urine lure, developed to target exophilic and exophagic malaria vectors outdoors, for which efficient surveillance and control tools are currently lacking. The efficacy of the lure was dependent on both dose and the placement of the traps used to capture select mosquito species, as demonstrated here. While additional testing of the lure in other geographic regions, with other landscape and vector species profiles, may be required, the main obstacles to be overcome include the high regulatory burden imposed on semiochemical tools used for integrated vector control. Moreover, this novel integrated vector management technology must be reviewed, evaluated, and recommended by the World Health Organization prior to being adopted by national programmes.
Availability of data and materials
All data generated and/or analysed during this study are included in this published article. The materials collected during the study are available from the corresponding author on reasonable request.
Generalized linear mixed effect model
Takken W, Knols BG. Malaria vector control: current and future strategies. Trends Parasitol. 2009;25:101–4.
Matowo NS, Moore J, Mapua S, Madumla EP, Moshi IR, Kaindoa EW, et al. Using a new odour–baited device to explore options for luring and killing outdoor–biting malaria vectors: a report on design and field evaluation of the mosquito landing box. Parasit Vectors. 2013;6:137.
Okumu FO, Sumaye RD, Matowo NS, Mwangungulu SP, Kaindoa EW, Moshi IR, et al. Outdoor mosquito control using odour–baited devices: development and evaluation of a potential new strategy to complement indoor malaria prevention methods. MalariaWorld J. 2013;4:8.
Reddy MR, Overgaard HJ, Abaga S, Reddy VP, Caccone A, Kiszewski AE, et al. Outdoor host seeking behaviour of Anopheles gambiae mosquitoes following initiation of malaria vector control on Bioko Island. Equatorial Guinea Malar J. 2011;10:184.
Russell TL, Govella NJ, Azizi S, Drakeley CJ, Kachur SP, Killeen GF. Increased proportions of outdoor feeding among residual malaria vector populations following increased use of insecticide–treated nets in rural Tanzania. Malar J. 2011;10:80.
Durnez L, Coosemans M. Residual transmission of malaria: an old issue for new approaches. In: Manguin S, editor. Anopheles mosquitoes: new insights into malaria vectors. London: Intech; 2013. p. 671–704.
Russell TL, Beebe NW, Cooper RD, Lobo NF, Burkot TR. Successful malaria elimination strategies require interventions that target changing vector behaviours. Malar J. 2013;12:56.
Kessy ST. A passive odor baited device for controlling Anopheles arabiensis mosquitoes. Dissertation for Award of MSc Degree at University of Dar es Salaam, Tanzania. 2016;1–108.
Sougoufara S, Ottih EC, Tripet F. The need for new vector control approaches targeting outdoor biting Anopheline malaria vector communities. Parasit Vectors. 2020;13:295.
Okumu FO, Killeen GF, Ogoma S, Biswaro L, Smallegange RC, Mbeyela E, et al. Development and field evaluation of a synthetic mosquito lure that is more attractive than humans. PLoS ONE. 2010;5:e8951.
Mukabana WR, Mweresa CK, Otieno B, Omusula P, Smallegange RC, Van Loon JJ, et al. A novel synthetic odorant blend for trapping of malaria and other African mosquito species. J Chem Ecol. 2012;38:235–44.
Sternberg ED, Waite JL, Thomas MB. Evaluating the efficacy of biological and conventional insecticides with the new ‘MCD bottle’ bioassay. Malar J. 2014;13:499.
Molleman F. Puddling: from natural history to understanding how it affects fitness. Entomol Exp Appl. 2010;134:107–13.
Webster B, Cardé RT. Use of habitat odour by host-seeking insects. Biol Rev Camb Philos Soc. 2017;92:1241–9.
Kwan MW, Bosak A, Kline J, Pita MA, Giel N, Pereira RM, et al. A low–cost, passive release device for the surveillance and control of mosquitoes. Int J Environ Res Public Health. 2019;16:1488.
Kweka EJ, Mwang’onde BJ, Kimaro E, Msangi S, Massenga CP, Mahande AM. A resting box for outdoor sampling of adult Anopheles arabiensis in rice irrigation schemes of lower Moshi, northern Tanzania. Malar J. 2009;8:82.
Kweka EJ, Owino EA, Mwang’onde BJ, Mahande AM, Nyindo M, Mosha F. The role of cow urine in the oviposition site preference of culicine and Anopheles mosquitoes. Parasit Vectors. 2011;4:184.
Mahande AM, Mwang’onde BJ, Msangi S, Kimaro E, Mnyone LL, Mazigo HD, et al. Is aging raw cattle urine efficient for sampling Anopheles arabiensis Patton? BMC Infect Dis. 2010;10:172.
Dawit M, Hill SR, Birgersson G, Tekie H, Ignell R. Malaria mosquitoes acquire and allocate cattle urine to enhance life history traits. Malar J. 2022;21:180.
Debebe Y. Studies on the spatial ecology of malaria and the impact of mass trapping of anopheles mosquitoes on malaria transmission in southern Ethiopia. Doctoral Thesis, Addis Ababa University, Ethiopia. 2020;1–108.
Balama C, Augustino S, Eriksen S, Makonda FS, Amanzi N. Climate change adaptation strategies by local farmers in Kilombero District. Tanzania Ethiop J Environ Stud Manag. 2013;6:724–36.
Finda MF, Limwagu AJ, Ngowo HS, Matowo NS, Swai JK, Kaindoa E, et al. Dramatic decreases of malaria transmission intensities in Ifakara, south–eastern Tanzania since early 2000s. Malar J. 2018;17:362.
Meza FC, Kreppel KS, Maliti DF, Mlwale AT, Mirzai N, Killeen GF, et al. Mosquito electrocuting traps for directly measuring biting rates and host-preferences of Anopheles arabiensis and Anopheles funestus outdoors. Malar J. 2019;18:83.
Harchut K, Standley C, Dobson A, Klaassen B, Rambaud-Althaus C, Althaus F, et al. Over–diagnosis of malaria by microscopy in the Kilombero Valley, Southern Tanzania: an evaluation of the utility and cost–effectiveness of rapid diagnostic tests. Malar J. 2013;12:159.
Team RC. R: A Language and Environment for Statistical Computing. R Found. Stat. Comput. 2016.
Wondwosen B, Birgersson G, Seyoum E, Tekie H, Torto B, Fillinger U, et al. Rice volatiles lure gravid malaria mosquitoes. Anopheles arabiensis Sci Rep. 2016;6:37930.
Gillies M, Meillon D. The Anophelinae of Africa south of the Sahara (Ethiopian Zoogeographical Region). Johannesburg: S Afr Inst Med Res. 1968. p. 343.
Gillies MT, Coetzee M. A supplement to the Anophelinae of Africa South of the Sahara. Publ S Afr Inst Med Res. 1987;55:1–43.
Cornel AJ, Porter CH, Collins FH. Polymerase chain reaction species diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences. J Med Entomol. 1996;33:109–16.
Hunt RH, Coetzee M, Fettene M. The Anopheles gambiae complex: a new species from Ethiopia. Trans R Soc. 1998;92:231–5.
Cohuet A, Simard F, Toto JC, Kengne P, Coetzee MA, Fontenille D. Species identification within the Anopheles funestus group of malaria vectors in Cameroon and evidence for a new species. Am J Trop Med Hyg. 2003;69:200–5.
Koekemoer LL, Kamau L, Hunt RH, Coetzee M. A cocktail polymerase chain reaction assay to identify members of the Anopheles funestus (Diptera: Culicidae) group. Am J Trop Med Hyg. 2002;66:804–11.
Zuur AF, Ieno EN, Elphick CS. A protocol for data exploration to avoid common statistical problems. Methods Ecol Evol. 2010;1:3–14.
Gillies MT. Studies of house leaving and outside resting of Anopheles gambiae Giles and Anopheles funestus Giles in east Africa. I. The outside resting population. Bull Ent Res. 1954;45:361–74.
Trawinski PR, Mackay DS. Identification of environmental covariates of West Nile virus vector mosquito population abundance. Vector Borne Zoonotic Dis. 2010;10:515–26.
Torr SJ. Dose responses of tsetse flies (Glossina) to carbon dioxide, acetone and octenol in the field. Physiol Entomol. 1990;15:93–103.
We thank the village leaders and the community of Sagamanganga for allowing us to work in their village, fields and households. We particularly thank Sambo Maganga, Nicolaus Mwakalinga, Mseti William, Arnold Mmbando, Halfan Ngowo, Emmanuel Kaindoa for their great technical support. Also, all volunteers who participated in the collection of mosquitoes, namely Shinje Giduka, Ngunda Elia, Kulwa Gwala, and Giloti Gwala are warmly thanked. The authors wish to especially thank the staff from IHI and NIMR Amani center and retired Mr. Myamba, J for their support during the project.
Open access funding provided by Swedish University of Agricultural Sciences. This work was supported by the Swedish Research Council by a grant to SRH (2017–05536).
Ethics approval and consent to participate
Written and signed consent was obtained from each household head in order to enroll his/her household in the study and access their private land. All volunteers were provided with consent forms, translated to local language (Kiswahili), to fill out before the study began. Ethical approval for the study was obtained from the Institutional Review Board of the Ifakara Health Institute (Ref. IHI/IRB/EXT/No: 23—2019) and the Medical Research Coordination Committee at the National Institute for Medical Research (Ref. NIMR/HQ/R.8a/Vol.IX/3085). The research team conducted meetings with local government administration officers and village leaders before the study was initiated in Sagamaganga. In these meetings, the objectives of the study, methods, and duration were discussed, and the staff of the study was introduced to the public.
Consent for publication
Permission to publish this work was also obtained from NIMR (Ref: NIMR/HQ/P.12 VOL XXXIV/68).
The authors declare that they have no competing interests.
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Katusi, G.C., Makayula, S.M., Govella, N.J. et al. Distance from human dwellings differentially affects the efficacy of a synthetic cattle urine odour lure to trap malaria vectors. Malar J 22, 8 (2023). https://doi.org/10.1186/s12936-022-04437-7