{"paper_id":"7ac687be-5c90-4f39-8f56-f5251bcb2c12","body_text":"1 \n \nInnovating Urban Mosquito Control: Introducing Human-Controlled Breeding Sites as a 1 \nComponent of Integrated Mosquito Management (IMM) 2 \nGuillermo A. Baigorria1,2 and Consuelo C. Romero1,2 3 \n1 Next Season Systems LLC, Department of Research and Development, Lincoln, NE 68506, USA 4 \n2 Gikarix SAC, Department of Research and Development. La Molina, Lima, 15024, Peru 5 \nABSTRACT: 6 \nThis study introduces a novel method for mosquito control tailored specifically for urban and 7 \nsuburban areas, addressing their unique challenges. The concept of Human-Controlled Breeding 8 \nSites (HCBS) is presented as an innovative approach to reducing mosquito populations by 9 \nproviding controlled oviposition sites within households. The paper is structured into three main 10 \ncomponents. The first details the meticulous development of the HCBS method, the second 11 \nexplores the intricate development of the HCBS device, crucial for effective implementation of 12 \nthe HCBS approach. Finally, the experiment validating the HCBS methodology is described, 13 \noffering insights into its practical application and efficacy in mosquito population control. Our 14 \nproposal emphasizes the importance of considering mosquito needs, behavior and preferences in 15 \ndesigning effective mosquito control strategies. Gravid mosquitoes must find a suitable place for 16 \noviposition, and HCBS provides an easy-access controlled environment for them to do so within 17 \nhouseholds. Contrary to the commonly current recommendation to destroy all possible locations 18 \nwhere mosquitoes can lay their eggs, the HCBS methodology provides an opportunity for gravid 19 \nmosquitoes to oviposit their eggs without being deterred by chemical odors or dead larvae or 20 \npupae. It involves waiting for a timespan before effectively collecting and destroying the 21 \nindividuals in one concerted effort with zero impact on the environment and at minimal cost. 22 \nSuccessful deposition of eggs by gravid mosquitoes into HCBS devices, followed by their 23 \ndestruction within the designated timeframe, validates the system's efficacy in disrupting the 24 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \nmosquito life cycle. Moreover, our findings demonstrate the significant influence of color 1 \nselection on HCBS effectiveness, with green HCBS devices attracting the highest number of 2 \ngravid female mosquitoes for oviposition. These integrated components offer a comprehensive 3 \nunderstanding of the HCBS approach, effectively bridging the gap between methodological 4 \ndevelopment, device design, and practical application. The findings underscore HCBS as a 5 \nvaluable addition to mosquito control strategies, with potential applications in diverse 6 \nenvironments. Further research is essential to delve into and model the long-term effectiveness of 7 \nHCBS, aiming to optimize its design for maximum efficacy and scalability. This includes 8 \nstudying its performance over extended periods and refining the design parameters to enhance its 9 \nfunctionality and widespread applicability including the use of disaster risk management 10 \nsystems. 11 \nKeywords: Mosquito control, Human-Controlled Breeding Sites (HCBS), Integrated Mosquito 12 \nManagement, Oviposition sites, Vector control methods, Gravid female mosquitoes, Mosquito life 13 \ncycle, Public health 14 \n1. INTRODUCTION: 15 \nClimate change has emerged as a significant driver of mosquito population dynamics, 16 \ncontributing to the expansion of mosquito habitats into new geographic regions (Mora, et al., 17 \n2022). Rising temperatures, altered precipitation patterns, and changes in humidity create 18 \nfavorable conditions for mosquito breeding and survival, facilitating their spread into previously 19 \nunaffected areas (Lafferty, 2009). As a result, regions that were once considered free from 20 \nmosquito-borne diseases are now at risk of transmission due to the establishment of mosquito 21 \npopulations in these newly suitable habitats. 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n3 \n \nUrbanization exacerbates the impact of climate change on mosquito-borne disease 1 \ntransmission by creating environments conducive to mosquito proliferation. Rapid urban growth, 2 \nparticularly in tropical and subtropical regions, leads to the development of densely populated 3 \nurban and suburban areas with abundant breeding sites for mosquitoes, such as discarded 4 \ncontainers, sewage systems, and stormwater drains (Ananya and Miller, 2021; Guagliardo et al., 5 \n2015). The convergence of increased human population density and expanding mosquito 6 \npopulations in urban settings creates ideal conditions for the transmission of diseases like 7 \nDengue fever, Malaria, West Nile virus and Zika virus. 8 \nTraditional mosquito control methods, primarily developed for rural environments with 9 \nlow population density, face significant challenges in urban and suburban areas. The widespread 10 \nuse of chemical insecticides may be impractical or environmentally undesirable in densely 11 \npopulated urban settings, while biological control methods may be less effective due to the 12 \nlimited availability of natural habitats for mosquito predators and parasites (Chadee et al., 2009). 13 \nMoreover, the heterogeneous and dynamic nature of urban landscapes poses challenges for 14 \nimplementing source reduction and habitat modification strategies. 15 \nAddressing the growing threat of mosquito-borne diseases in urban and suburban areas 16 \nrequires the development of innovative and context-specific control methods tailored to urban 17 \nenvironments. Integrated approaches that combine novel vector control technologies with 18 \ncommunity engagement and public health interventions offer promising solutions for mitigating 19 \ndisease transmission in urban settings (Muller et al., 2020). These approaches may include the 20 \nuse of novel larvicides targeting mosquito breeding sites in urban landscapes, deployment of 21 \nspatial repellents to protect high-risk populations, and implementation of community-based 22 \nsurveillance and response systems to detect and control disease outbreaks. 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n4 \n \nEfforts to develop and implement new mosquito control methods for urban and suburban 1 \nconditions must consider the social, economic, and environmental factors that influence disease 2 \ntransmission dynamics in these settings (Bazin and Williams, 2018). Multidisciplinary 3 \ncollaborations between researchers, policymakers, public health authorities, and communities are 4 \nessential for designing effective and sustainable strategies to reduce the burden of mosquito-5 \nborne diseases in urban areas. 6 \nThe objectives of the present study are: 7 \n1. To introduce a novel method for mosquito control specifically designed for urban and 8 \nsuburban areas, addressing the unique challenges these environments present. 9 \n2. To present the concept of Human-Controlled Breeding Sites (HCBS) as a new, innovative 10 \napproach to mosquito population reduction by providing a controlled environment for 11 \nmosquito females to oviposit their eggs within households. 12 \n3. To describe the design and functionality of the Human-Controlled Breeding Site device, 13 \nemphasizing its potential as a supplementary component of Integrated Mosquito 14 \nManagement (IMM) strategies. 15 \n4. To investigate the oviposition preferences of mosquito females by testing multiple colors 16 \nwithin the Human-Controlled Breeding Sites devices, aiming to identify the most 17 \nattractive color for egg-laying and optimize the effectiveness of the control method. 18 \nThis paper is structured into three main components. In Section 3.1, we detail the 19 \nmeticulous development of the new HCBS method, laying the foundational groundwork for 20 \nsubstantiating the subsequent exploration and validation of the HCBS methodology. Section 3.2 21 \ndelves into the intricate development of the HCBS device, an essential component for 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n5 \n \nimplementing the HCBS approach effectively. Finally, Section 3.3 is dedicated to describing the 1 \nexperiment conducted to validate the HCBS methodology, offering insights into its practical 2 \napplication and efficacy in mosquito population control. Together, these integrated components 3 \nprovide a comprehensive understanding of the HCBS approach, effectively bridging the gap 4 \nbetween methodological development, device design, and practical application. 5 \n2. LITERATURE REVIEW: 6 \nMosquito-borne diseases pose significant threats to public health, particularly in tropical 7 \nand subtropical regions worldwide. Among the most notorious mosquito-borne illnesses are 8 \nDengue fever, Malaria, Yellow Fever, West Nile virus and Zika, which collectively cause 9 \nmillions of cases and thousands of deaths each year (World Health Organization, 2020). 10 \nControlling the populations of disease-carrying mosquitoes is crucial for reducing the 11 \ntransmission and burden of these diseases. In this literature review, we explore the biology and 12 \nbehavior of mosquitoes, existing control methods, factors influencing mosquito attraction, and 13 \nthe relationship between mosquito species and disease transmission. 14 \n2.1 Mosquito Biology and Behavior 15 \nMosquitoes, belonging to the Culicidae family, are small, flying insects characterized by 16 \ntheir slender bodies, long legs, and elongated mouthparts adapted for piercing and sucking blood 17 \n(Clements, 1999). The life cycle of mosquitoes consists of four stages: egg, larva, pupa, and 18 \nadult. 19 \nMale and female mosquitoes exhibit significant differences in their lifespans. Male 20 \nmosquitoes typically live for up to 10 days (Clements, 1999, Lambert et. al. 2022), while females 21 \ncan survive for a few weeks (Foster and Walker, 2009). Some sources suggest that males live for 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n6 \n \n1-2 weeks (Service, 2012), and females typically live anywhere from two to four weeks (Scott 1 \nand Takken, 2012). 2 \nThe primary reason for this difference in lifespan is the distinct roles each gender plays 3 \nwithin the species. Males primarily live to mate and do not play a major role in the reproductive 4 \ncycle after mating (Service, 2012). On the other hand, female mosquitoes require extended 5 \nperiods of life to lay eggs (Clements, 1999). 6 \nMoreover, the feeding habits of male and female mosquitoes also contribute to their 7 \nlifespan differences. Male mosquitoes feed only on plant nectar and sugary liquids, such as fruit 8 \njuices and honeydew (Clements, 1999). In contrast, female mosquitoes feed on blood in addition 9 \nto plant nectar, which provides the necessary nutrients for egg production (Foster and Walker, 10 \n2009). 11 \nTherefore, the differences in lifespan between male and female mosquitoes can be 12 \nattributed to their distinct roles in reproduction and their different feeding habits (Clements, 13 \n1999; Foster and Walker, 2009; Service, 2012; Scott and Takken, 2012), although a new study 14 \nsuggest the lifespan difference can be less (Lambert et al., 2022). 15 \nEnvironmental factors significantly influence mosquito biology and behavior, with 16 \ntemperature, humidity, and the availability of breeding sites playing crucial roles in shaping 17 \nmosquito population dynamics and distribution (Hugo et al., 2014; Paaijmans et al., 2009). 18 \nHuman-related factors, such as urbanization (Durrance-Bagale et al., 2024) and changes in land 19 \nuse, can also have a substantial impact on mosquito habitats and abundance (Gubler, 2011; 20 \nLeisnham et al., 2014). Additionally, characteristics like water type (Sumba et al., 2008), larvae 21 \ndensity (Allan and Kline, 1998; Gimnig et al., 2002; Kiflawi et al., 2003; Koenraadt and Takken, 22 \n2003), and even pheromone emissions by conspecific larvae (Chadee, 1993; Sumba et al., 2008) 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n7 \n \ninfluence oviposition behavior. However, a common thread among these factors is the necessity 1 \nof water in all oviposition sites. 2 \n2.2 Mosquito Control Methods 3 \nThe World Health Organization (WHO, 2009) advocates for integrated vector 4 \nmanagement (IVM) and suggests four major strategies: Chemical control involves the use of 5 \ninsecticides to kill mosquito larvae and adults. These variously comprise fogging, residual 6 \nspraying, larviciding, and autodissemination. However, the emergence of insecticide resistance 7 \nposes challenges to the effectiveness of chemical control efforts (Hemingway and Ranson, 8 \n2000). 9 \nBiological control methods harness natural enemies of mosquitoes, such as predators and 10 \nparasites, to reduce mosquito populations. Predatory fish, such as Gambusia affinis (mosquito 11 \nfish), feed on mosquito larvae and are used in biological control programs (Sota and Mogi, 12 \n1992). Additionally, genetically modified mosquitoes are being developed as a novel approach to 13 \nsuppress mosquito populations (Alphey et al., 2002). 14 \nEnvironmental management strategies focus on reducing mosquito breeding sites and 15 \nmodifying habitats to make them less suitable for mosquito development. Source reduction 16 \ntechniques involve eliminating or treating standing water where mosquitoes breed, while habitat 17 \nmodification aims to alter environmental conditions to discourage mosquito proliferation 18 \n(Heintze et al., 2007).  19 \nPersonal barrier measures involve using barriers like window screens, mosquito nets, 20 \nrepellents, or protective clothing to prevent mosquito bites. 21 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n8 \n \nIntegrated Mosquito Management (IMM) emphasizes the coordinated use of multiple 1 \ncontrol methods tailored to local conditions. Surveillance, larval control, adult control, and 2 \ncommunity engagement are key components of IMM programs aimed at reducing mosquito 3 \npopulations and disease transmission (World Health Organization, 2019). 4 \n2.3 Mosquito Attraction and Repellents 5 \nMosquitoes are attracted to their hosts by a combination of visual and chemical cues. 6 \nVisual cues, such as color, play a role in mosquito attraction. Research suggests that mosquitoes 7 \nare more attracted to dark colors, possibly due to their increased visibility against contrasting 8 \nbackgrounds (Foster and Hancock, 1994). 9 \nChemical cues, including carbon dioxide (CO2) and human body odors, also influence 10 \nmosquito host-seeking behavior. Mosquitoes are highly sensitive to CO2, which is emitted by 11 \nhumans during respiration and serves as a potent attractant for female mosquitoes seeking blood 12 \nmeals (Takken and Knols, 1999). Additionally, human skin odors, such as lactic acid and 13 \nammonia, can attract mosquitoes from a distance (Logan et al., 2008). 14 \nVarious repellents are available to protect against mosquito bites. Synthetic chemicals, 15 \nsuch as DEET (N,N-diethyl-meta-toluamide), are widely used and highly effective at repelling 16 \nmosquitoes (Frances and Cooper, 2005). Plant-derived repellents, such as citronella and 17 \neucalyptus oil, offer alternative options for personal protection against mosquito bites (Maia and 18 \nMoore, 2011). 19 \n2.4 Mosquito Species and Disease Transmission 20 \nDifferent mosquito species exhibit varying degrees of vector competence for transmitting 21 \ndiseases to humans. Aedes aegypti and Aedes albopictus are primary vectors of Dengue fever, 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n9 \n \nZika virus, West Nile virus and Chikungunya virus, with Aedes aegypti being particularly 1 \nefficient in urban environments (Lambrechts et al., 2010). Anopheles mosquitoes are the main 2 \nvectors of Malaria, transmitting Plasmodium parasites to humans through their blood meals 3 \n(Sinka et al., 2012). 4 \nThe relationship between mosquito species and disease transmission is influenced by 5 \nfactors such as vector biology, host preferences, and pathogen development. Understanding the 6 \necology and behavior of mosquito vectors is essential for implementing targeted control 7 \nmeasures to reduce disease transmission (Weiss et al., 2019). 8 \n2.5 Mosquito's oviposition ethology 9 \nMosquito oviposition behavior, influenced by various factors including color preferences 10 \nand environmental cues, plays a crucial role in the transmission of diseases. Different mosquito 11 \nspecies exhibit distinct preferences in color for oviposition sites, often correlating with the 12 \ndiseases they transmit. Understanding these ethological preferences is essential for effective 13 \nvector control strategies and disease prevention efforts. 14 \n2.5.1 Aedes aegypti: 15 \nAedes aegypti is a primary vector of several devastating diseases, including dengue fever, 16 \nZika virus, West Nile virus, and chikungunya. Research has shown that Aedes aegypti females 17 \nexhibit a preference for dark colors such as black and brown for oviposition sites (Bentley and 18 \nDay, 1989). These dark-colored sites often contain stagnant water, such as discarded containers, 19 \nwhich serve as breeding grounds for Aedes mosquitoes and contribute to the transmission of 20 \ndiseases like dengue fever and Zika virus. 21 \n2.5.2 Aedes albopictus: 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n10 \n \nAnother important vector species, Aedes albopictus, is associated with the transmission 1 \nof diseases such as dengue fever, Zika virus, West Nile virus, and chikungunya. Unlike Aedes 2 \naegypti, Aedes albopictus displays a preference for lighter colors like white or light blue for 3 \noviposition (Koenraadt et al., 2003). These preferences may vary depending on environmental 4 \nconditions and habitat characteristics, influencing the distribution of oviposition sites and disease 5 \ntransmission patterns. 6 \n2.5.3 Culex pipiens: 7 \nCulex pipiens is a widespread mosquito species known for transmitting West Nile virus, a 8 \npotentially severe neurological disease. Studies have indicated that Culex pipiens females prefer 9 \noviposition sites with high organic content, often found in dark-colored water bodies (Rey et al., 10 \n2006). These preferences contribute to the proliferation of Culex mosquitoes and the 11 \ntransmission of West Nile virus to humans and other vertebrate hosts. 12 \n2.5.4 Anopheles spp.: 13 \nAnopheles mosquitoes are vectors of malaria parasites, a life-threatening disease that 14 \naffects millions of people worldwide. Different species within the Anopheles genus may exhibit 15 \nvarying oviposition preferences. For example, Anopheles gambiae, a major malaria vector in 16 \nsub-Saharan Africa, shows a preference for dark-colored oviposition sites (Munga et al., 2006). 17 \nThese sites, often found in natural water bodies like swamps and puddles, provide suitable 18 \nbreeding habitats for Anopheles mosquitoes and contribute to the transmission of malaria. 19 \nIn addition to color preferences, environmental factors such as temperature, humidity, and 20 \nvegetation also influence mosquito oviposition behavior and disease transmission dynamics. 21 \nMosquitoes prefer shaded areas with moderate temperatures and high humidity for oviposition, 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n11 \n \nas these conditions promote larval survival and development (Lounibos, 2002). Vegetation 1 \nprovides additional shelter and resources, creating ideal habitats for mosquito breeding and 2 \ndisease transmission (Clements, 1999). 3 \n2.5.5 Oviposition radius 4 \nGravid mosquito females exhibit a remarkable ability to search for suitable oviposition 5 \nsites, often traveling significant distances and enduring varying environmental conditions to lay 6 \ntheir eggs in standing water. The duration and distance traveled by gravid females largely depend 7 \non factors such as species, habitat availability, and physiological state. For instance, Aedes 8 \nmosquitoes, vectors of diseases like dengue and Zika, have been observed to travel up to several 9 \nhundred meters in search of suitable breeding sites, with some individuals capable of flying 10 \ndistances of over a kilometer (Service, 1997). Similarly, Anopheles mosquitoes, responsible for 11 \ntransmitting malaria, are known to actively seek out water bodies for oviposition, often traveling 12 \nseveral kilometers from their blood-feeding sites (Clements, 1999). Gravid females may spend 13 \nseveral days or even weeks in search of suitable oviposition sites, utilizing various sensory cues 14 \nand environmental cues to guide their behavior (Clements, 1999). This remarkable behavior 15 \nunderscores the importance of effective mosquito control strategies to target breeding sites and 16 \nminimize the risk of disease transmission. 17 \n2.5.6 Mosquito eggs viability 18 \nMosquito eggs possess remarkable resilience, capable of surviving in a desiccated state 19 \nfor extended periods. Research indicates that mosquito eggs can remain viable for varying 20 \ndurations without water, depending on species and environmental conditions. For instance, 21 \nAedes mosquitoes, known vectors of diseases such as dengue and Zika, have been reported to 22 \nmaintain viability for up to several months under favorable conditions (Christophers, 1960). 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n12 \n \nSimilarly, studies on Anopheles mosquitoes, responsible for transmitting malaria, suggest that 1 \ntheir eggs can endure desiccation for several weeks, retaining the potential for hatching upon 2 \nrehydration (Service, 1997). This adaptive trait enables mosquito eggs to persist through adverse 3 \nenvironmental conditions, contributing to the resilience and persistence of mosquito populations. 4 \nMosquito eggs are typically laid on the surface of standing water, although some species 5 \nmay deposit them on moist substrates near water bodies. The surface location of mosquito eggs 6 \nserves several crucial functions in the reproductive process. Firstly, positioning the eggs on the 7 \nwater surface provides protection from drowning and predation, as it allows for easy access to 8 \natmospheric oxygen for respiration (Clements, 1999). Additionally, the surface tension of the 9 \nwater supports the buoyancy of the eggs, preventing them from sinking and facilitating their 10 \ndevelopment (Clements, 2000). 11 \nThe exchange of oxygen is vital for the survival and development of mosquito eggs. Like 12 \nall insect eggs, mosquito eggs require oxygen for respiration during embryonic development. 13 \nThe thin outer layer of the egg, called the chorion, is permeable to gases, allowing oxygen to 14 \ndiffuse into the egg while carbon dioxide is released (Clements, 1999). This exchange of gases 15 \nthrough the eggshell is essential for maintaining aerobic respiration and ensuring the viability of 16 \nthe developing embryo. 17 \nVarious household substances have been identified for their efficacy in destroying 18 \nmosquito eggs, ranging from natural and environmentally friendly options to chemical 19 \ncompounds with larvicidal properties. Soap disrupts water surface tension, suffocating mosquito 20 \neggs, while vinegar creates an inhospitable acidic environment hindering egg development. 21 \nEssential oils like citronella possess larvicidal properties, deterring egg-laying and destroying 22 \nexisting eggs, and diluted bleach solutions disrupt larvae and egg integrity. Additionally, a thin 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n13 \n \nlayer of vegetable oil forms a barrier on water surfaces, suffocating eggs. However, effectiveness 1 \ncan vary based on factors like concentration and application method, emphasizing the need for 2 \ncaution, especially with chemical-based products, to ensure safety for humans and non-target 3 \norganisms. (Rajapaksa et al., 2014; Clements, 1999; Maia and Moore, 2011) 4 \n3. MATERIALS AND METHODS: 5 \n3.1 The Human-Controlled Breeding Site (HBCS) method 6 \nThe HCBS method represents a proactive approach to controlling mosquito populations 7 \nthrough targeted intervention at key stages of the mosquito life cycle. This section delineates the 8 \nstep-by-step methodology for implementing the HCBS method, emphasizing its efficacy in 9 \nattracting gravid female mosquitoes and disrupting the mosquito reproductive process. 10 \n3.1.1 Deployment of HCBS Devices 11 \nThe first step in the HCBS method involves the strategic placement of HCBS devices in 12 \nareas susceptible to mosquito breeding in urban and suburban environments. HCBS devices are 13 \npositioned at floor level to maximize visibility to flying female mosquitoes while remaining 14 \ninconspicuous to human observation. The devices are distributed evenly across the target area to 15 \nensure comprehensive coverage and efficacy in mosquito population control. 16 \nTo minimize breeding site competition effectively, two strategies are essential: (a) the 17 \nelimination of existing breeding sites, and/or (b) the incorporation of other standing water 18 \nsources into the HCBS system. In the former approach, the nearest breeding site would be the 19 \nHCBS device, compelling gravid mosquitoes to utilize it exclusively. Conversely, in the latter 20 \napproach, regulating potential standing water sources to align with the HCBS methodology 21 \ntransforms them into functional HCBS devices. 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n14 \n \n3.1.2 Attracting Gravid Female Mosquitoes 1 \nOnce installed, HCBS devices are designed to attract gravid female mosquitoes seeking 2 \noviposition sites for eggs that must be laid. The design features of the HCBS devices, including a 3 \nshallow basin and a portion of the water surface covered from light, mimic natural breeding 4 \nhabitats favored by mosquitoes. This strategic design encourages female mosquitoes to lay their 5 \neggs in the HCBS devices, thereby initiating the mosquito life cycle within a controlled 6 \nenvironment. 7 \n3.1.3 Removal and Treatment of Water 8 \nAfter a predetermined period, typically 10 days to coincide with the development of 9 \nmosquito larvae into pupae, the water from the HCBS devices is carefully removed and 10 \ntransferred to a separate container. This step is crucial since if the water is not removed within 11 \nthis timeframe, mosquito pupae will emerge as adults, rendering the method ineffective and 12 \ninadvertently contributing to mosquito reproduction. Moving forward, it is imperative to explore 13 \nthe feasibility of automating the water replacement process in HCBS devices to mitigate this 14 \nissue and ensure the continued efficacy of mosquito population control efforts. 15 \n3.1.4 Destroying and killing the collected eggs, larvae and pupas. 16 \nOne option to destroy and kill the eggs, larvae and pupas from the HCBS devices, it’s to 17 \ntreat it with household substances known for their larvicidal properties. Common substances 18 \nsuch as soap, vinegar, essential oils, or bleach are added to the water to destroy mosquito eggs, 19 \nlarvae, and pupae present in the container. This targeted treatment ensures the eradication of 20 \nmosquito offspring and prevents their proliferation in the environment (see Section 2.5.5). 21 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n15 \n \nIt is crucial to emphasize that only water should be poured into the HCBS container. If 1 \nthe mosquito female detects traces of other substances, including insecticides or household 2 \nproducts discussed previously, there is a risk that she may reject the HCBS device as an 3 \noviposition site. This would render the method ineffective and unsuitable for mosquito 4 \npopulation control. 5 \nIn dire scenarios where household substances are unavailable, simply pouring the water 6 \nfrom the HCBS device onto a dry surface can effectively eliminate larvae and pupae but not 7 \nnecessary eggs. However, the easiest alternative to kill the individuals on the three stages is just 8 \nto bury the collected water beneath a few centimeters of soil (in a garden or even a pot), thus 9 \nensuring continued mosquito population control. 10 \n3.1.5 Reinstallation of HCBS Devices 11 \nFollowing treatment, the HCBS devices are refilled with fresh water and reinstated in 12 \ntheir original location. 13 \n3.2 Considerations taken during the design of the HCBS Device 14 \nThe design of the Human-Controlled Breeding Site (HCBS) device was informed by a 15 \ncomprehensive literature review on mosquito ethology and its interactions with humans (Section 16 \n2). Understanding the behavior of mosquitoes and their preferences for oviposition sites needs 17 \nwas crucial in developing an effective breeding site for controlling mosquito populations. 18 \n3.2.1 Shape Design 19 \nThe HCBS device was designed with a shallow basin to mimic natural breeding habitats 20 \nfavored by mosquitoes. Its shape allows for a relatively large-exposed water surface, providing 21 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n16 \n \nample space for egg deposition. Additionally, the device incorporates a portion of the water 1 \nsurface covered from light, offering protection to mosquito offspring against potential predators. 2 \n3.2.2 Color Selection 3 \nFive colors were selected for testing the HCBS device: White, Black, Red, Green, and 4 \nBlue. Color selection played a significant role in determining the attractiveness of the device to 5 \ngravid female mosquitoes. The color of the HCBS device was chosen to ensure visibility to 6 \nmosquito females while remaining semi-hidden to humans. This balance is crucial to ensure that 7 \nmosquitoes perceive the oviposition site as hidden from predators. 8 \n3.2.3 Placement and Visibility 9 \nThe HCBS device is ideally located at floor level to ensure maximum visibility from 10 \nflying female mosquitoes seeking oviposition sites. It must be strategically positioned to be 11 \nvisible to mosquito females while remaining semi-hidden from human view, creating the 12 \nperception of a secure oviposition place away from predators. 13 \n3.2.4 Additional Features 14 \nTo facilitate transportation and maintenance, the HCBS device is equipped with a cap or 15 \nslicing cover to close the exposed water surface when not in use or during harvesting. This 16 \nfeature ensures the integrity of the breeding site and prevents contamination. Importantly, no 17 \ninsecticides or mosquito-killing devices are added to the water within the HCBS to avoid 18 \ndeterring gravid female mosquitoes from ovipositing. 19 \n3.3 Experiment design 20 \nThe experiment was conducted in Peru, specifically in the department of Lima, within the 21 \ndistrict of La Molina, which is situated at an altitude of approximately 237 meters above sea 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n17 \n \nlevel. Its geographical coordinates are approximately 12.0760° S latitude and 76.9502° W 1 \nlongitude. This region experiences climatic conditions typical of a coastal desert, characterized 2 \nby moderate temperatures, high relative humidity, and minimal rainfall. During the months of 3 \nFebruary to May, which coincide with the austral summer, La Molina typically records average 4 \ntemperatures ranging from 22°C to 27°C. Relative humidity levels vary between 60% to 70%. 5 \nRainfall is scarce, with an average total annual precipitation of less than 10 millimeters. 6 \nThe mosquito species commonly found in this area include Aedes aegypti, the primary 7 \nvector for diseases such as dengue, Zika, and chikungunya. Additionally, Culex quinquefasciatus, 8 \nknown for transmitting West Nile virus, and Anopheles species responsible for malaria 9 \ntransmission, are also prevalent in the region. 10 \nThe experiment was designed to evaluate the efficacy of Human-Controlled Breeding 11 \nSite (HCBS) devices in attracting gravid female mosquitoes for oviposition, with a focus on 12 \ncolor preference. The HCBS devices were specially designed based on findings from the 13 \nliterature review on mosquito ethology and breeding site preferences. Using 3D printing 14 \ntechnology, the devices were fabricated in ABS material in five different colors: white, black, 15 \nred, green, and blue. 16 \n3.3.1 Installation and Setup 17 \nAll HCBS devices were filled to the middle with potable water and installed in the same 18 \nlocation, following the specified placement guidelines to ensure maximum visibility to flying 19 \nfemale mosquitoes. After 10 days of exposure to mosquito access, the HCBS devices were 20 \ncapped and removed from their location. To minimize potential biases due to the very specific 21 \nlocation of each HCBS device within the installation location, the distribution of colors among 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n18 \n \nHCBS devices was randomly altered at each reinstallation. This process was replicated five times 1 \nto ensure robustness and reliability of the findings. 2 \n3.3.2 Data Collection and Analysis 3 \nIn the laboratory, a count of larvae and pupae from each HCBS device was conducted to 4 \nevaluate mosquito attraction. Due to equipment constraints, egg counts were not feasible for this 5 \nexperiment, and therefore, only larvae and pupae were included in the tally. After counting, the 6 \nHCBS devices were refilled with water and reinstalled in their original location. 7 \nIt's worth noting that in our study, we prioritized assessing the functionality of HCBS 8 \nrather than eradicating the mosquito population in our test area. Consequently, the eggs, larvae, 9 \nand pupae were not eradicated after collection; instead, they were transferred to another 10 \ncontainer to mature into adults and contribute to the ongoing mosquito population in the study 11 \narea. This approach ensured a consistent supply of adult mosquitoes for testing the effectiveness 12 \nof HCBS methodology and devices. 13 \n3.3.3 Laboratory Environment 14 \nThe installation site was situated inside a laboratory exposed to mosquito access, 15 \nproviding a semi-controlled environment for the experiment (conditions similar to those of a 16 \nroom in a house). This setting allowed for consistent conditions across replicates and minimized 17 \nexternal variables that could influence mosquito behavior and oviposition decisions. 18 \n4. RESULTS AND DISCUSSION: 19 \n4.1 The design  20 \nFigure 1 shows the final design of the HCBS device used in our study whereas Figure 2 21 \nshows the different color devices installed during the experiment. 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n19 \n \n 1 \n 2 \nFigure 1: Design drawings of the HCBS device, showing (a) top view of the body, (b) side view 3 \nof the body, (c) front view of the body, and (d) top view of the slicing cap. Measurements are in 4 \nmillimeters (mm). 5 \nThe slicing cap (d) is placed on the top view of the body (a), enabling slicing between 6 \nboth arms for easy opening and closing of the HCBS device. 7 \n 8 \n   \n   \n  \n     \n    \n   \n      \n    \n   \n  \n                                     \n   \n   \n   \n   \n                                   \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n20 \n \n 1 \nFigure 2: Photograph depicting HCBS devices in all colors used during the experiment, 2 \ninstalled in their respective locations. 3 \n4.2 Experiment results 4 \n4.2.1 Results on the HCBS method 5 \nThe successful deposition of eggs by gravid mosquitoes into the HCBS devices, followed 6 \nby the destruction of these individuals within the 10-day timeframe, serves as compelling 7 \nevidence of the efficacy of the system. This crucial demonstration underscores the functionality 8 \nof both the HCBS method and device. Without the presence of HCBS devices, these eggs would 9 \nhave inevitably hatched, contributing to the burgeoning mosquito population. Thus, the ability to 10 \nattract gravid mosquitoes and disrupt their reproductive cycle within the designated timeframe 11 \nserves as a robust validation of the HCBS system's effectiveness. This innovative approach 12 \nchallenges the commonly current recommendation to destroy all possible locations where 13 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n21 \n \nmosquitoes can lay their eggs, providing a controlled environment for oviposition within 1 \nhouseholds while simultaneously reducing mosquito populations at the source. 2 \n4.2.2 Results on the HCBS color device 3 \nThe sampled adult mosquito population from the laboratory revealed that all captured 4 \nmosquitoes belonged to the Aedes aegypti species.  5 \nTable 1: Number of mosquito’s larvae and pupas in the HCBS devices by color and date: 6 \nHCBS \ndevice color \nHarvesting date \nApril 20th April 30th May 10th May 20th May 30th \nBlack 0 0 0 0 0 \nBlue 0 0 0 0 0 \nGreen 2 8 17 15 12 \nRed 0 0 0 0 0 \nWhite 0 0 0 0 0 \n 7 \nAfter tallying the larvae and pupae across all HCBS devices, a significant statistical 8 \ndifference emerged in the number of specimens collected, particularly between the green HCBS 9 \ndevice and others of varying colors. This statistical difference arises from the exclusive 10 \nutilization of the green device by mosquitoes for oviposition, as outlined in Table 1. 11 \nContrastingly, the remaining colors did not serve as oviposition sites, leading to a lack of 12 \nstatistical difference among them. Furthermore, the temporal variation in the number of 13 \nindividuals within HCBS devices was influenced by declining temperatures observed during the 14 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n22 \n \nfinal assessment period, impacting not only our laboratory's mosquito population but also that of 1 \nthe broader region. 2 \nThe Human-Controlled Breeding Sites (HCBS) method demonstrates remarkable efficacy 3 \nin controlling mosquito populations, exerting influence over three of the four life stages of 4 \nmosquitoes. Our findings confirm that when HCBS is correctly applied, it achieves a 100% 5 \neffectiveness in disrupting the mosquito life cycle by collecting laid egg and eliminating all 6 \ntogether with larvae and pupae previously hatched. 7 \nOne of the key advantages of HCBS is its low implementation cost, which promotes 8 \nwidespread citizen participation in mosquito reduction campaigns. This cost-effectiveness not 9 \nonly encourages community engagement but also facilitates the scalability of mosquito control 10 \nefforts in urban and suburban areas. 11 \nIn the event of massive HCBS deployment in urban and suburban environments, a 12 \nsignificant reduction in mosquito populations can be anticipated. By disrupting reproduction, 13 \nHCBS ensures that the current adult mosquito population will be the last local generation. Within 14 \na short timeframe, typically within 1 week to 10 days, the absence of male mosquitoes prevents 15 \nfertilization, effectively limiting the population's ability to replenish since only unfertilized 16 \nfemale mosquitoes would theoretically remain alive after this period. 17 \nWhile the primary focus of HCBS implementation is in urban and suburban areas, its 18 \npotential applicability in rural settings warrants consideration. Gravid female mosquitoes tend to 19 \nseek the closest and safest place to lay their eggs. With HCBS devices installed within or around 20 \nhouses, mosquitoes are likely to prefer ovipositing in the HCBS rather than expending energy to 21 \nfind distant natural environments where predators exist (see Section 2.5.5). Although its 22 \neffectiveness may be somewhat diminished compared to urban areas, HCBS still serves to reduce 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n23 \n \nlocal mosquito populations and disrupt the feedback loop of mosquito repopulation in natural 1 \nenvironments. 2 \nOur study also identifies the importance of device color selection in optimizing HCBS 3 \neffectiveness. Among the colors tested, green HCBS devices consistently demonstrated the 4 \nhighest efficacy in attracting gravid female mosquitoes for oviposition. Additionally, HCBS 5 \ndevices must have a portion of the water surface shielded from light. This feature makes female 6 \nmosquitoes more likely to select this device for oviposition, as it offers protection for their 7 \noffspring against predators. This issue was verified during the retrieval and counting of the 8 \nindividuals collected in the laboratory. When the slicing cap of the HCBS device was opened 9 \nunder the lights, all larvae swiftly moved towards the shaded water within the device. This 10 \nfinding underscores the importance of considering mosquito behavior and preferences in the 11 \ndesign of effective mosquito control strategies. 12 \nOverall, the findings of this study underscore the potential of HCBS as a valuable 13 \naddition to Integrated Mosquito Management strategies, particularly in urban and suburban 14 \nsettings. By targeting multiple life stages of mosquitoes and incorporating features that enhance 15 \nattractiveness and efficacy, HCBS offers a promising approach to reducing mosquito populations 16 \nand mitigating the transmission of mosquito-borne diseases in diverse environments. Further 17 \nresearch and field trials are warranted to explore the long-term effectiveness and scalability of 18 \nHCBS implementation in different geographic contexts. Additionally, optimizing the design of 19 \nHCBS devices warrants attention to maximize their efficacy and applicability. Finally, the HCBS 20 \nmethod can undergo evaluation for potential application in combating other insect-borne vector 21 \ndiseases. 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n24 \n \nOne suggestion is to leverage disaster risk management systems capable of widespread 1 \ncommunication via SMS text messages to convey crucial information to residents in a specific 2 \narea. The proposal involves utilizing a country's system to coordinate the simultaneous 3 \ninstallation of HCBS devices across the entire community or city. Similarly, on the 10th day, 4 \nresidents would receive alerts prompting them to harvest and reinstall the HCBS devices. This 5 \napproach ensures uniform implementation of control programs throughout the community, 6 \nreminding residents simultaneously to remove the devices to mitigate the negative effects 7 \ndiscussed in Section 3.3.3. 8 \n5. CONCLUSIONS 9 \nThe Human-Controlled Breeding Site (HCBS) methodology is a groundbreaking 10 \napproach that diverges from the common recommendation to eliminate all potential mosquito 11 \negg-laying sites. Instead, it creates controlled breeding sites to attract female mosquitoes for 12 \noviposition. Since humans oversee these sites, the eggs, larvae, and pupae can be effectively 13 \nbeing mechanically destroyed before maturing into adult mosquitoes. HCBS proves to be a 14 \nhighly effective method for controlling mosquito populations due to its ability to intervene in 15 \nmultiple stages of the mosquito life cycle. By targeting three out of the four stages of mosquito 16 \ndevelopment, HCBS offers a comprehensive approach to mosquito control that can significantly 17 \nreduce mosquito populations and mitigate disease transmission in urban and suburban areas. 18 \nHCBS provides a controlled environment within households where mosquito females can 19 \noviposit their eggs. By attracting gravid females to lay their eggs in the breeding site, HCBS 20 \ndevises effectively intercepts the first stage of mosquito development, preventing eggs from 21 \nbeing laid in natural breeding habitats. Considering that the mosquito’s life cycle in a water 22 \nenvironment from egg to pupa has a timespan of around 10 days, at the time the HCBS is 23 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n25 \n \nharvested, it will contain individuals in all the three stages from eggs to pupas. At the time the 1 \nwater is discharged from the HCBS device, all the individuals collected are killed at once. 2 \nAlthough HCBS primarily targets the egg, larval, and pupal stages of the mosquito life 3 \ncycle, it complements existing mosquito control methods that focus on adult mosquitoes. By 4 \nintervening in multiple stages of mosquito development, HCBS provides a holistic approach to 5 \nmosquito control that enhances the effectiveness of Integrated Mosquito Management (IMM) 6 \nstrategies. Additionally, the localized nature of HCBS allows for targeted control efforts in areas 7 \nwith high mosquito abundance or disease transmission, contributing to a more efficient and 8 \nsustainable mosquitoes control programs. 9 \nThe HCBS device developed in this study played a pivotal role in facilitating the 10 \nscientific experimentation of the HCBS method, offering insights into critical factors such as 11 \ncolor selection for enhancing system efficiency. However, the paramount aspect lies in the 12 \nadaptability of the HCBS methodology, as any household receptacle capable of retaining water 13 \nfor over 10 days can serve as an HCBS device (e.g., cups, big-mouth bottles, dishes, etc.). When 14 \nconsidering alternative household HCBS devices, key considerations include color, shading, and 15 \ntotal water surface area exposed to attract gravid mosquitoes for oviposition. It is important to 16 \nremember that eggs must be laid, and gravid mosquitoes require standing water for this purpose, 17 \nwith no preferred options being available for their reproduction, any source will do. If we control 18 \nthe source, then we control the reproduction of those mosquitoes. 19 \nIn dire scenarios where household substances are unavailable, simply pouring the water 20 \nfrom the HCBS device onto a dry surface can effectively eliminate larvae and pupae but not 21 \nnecessary eggs. However, the easiest alternative to kill the individuals on the three stages is just 22 \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted May 14, 2024. ; https://doi.org/10.1101/2024.05.14.24307332doi: medRxiv preprint \n\n26 \n \nto bury the collected water beneath a few centimeters of soil in a garden or pot, ensuring 1 \ncontinued mosquito population control. 2 \nIn conclusion, this study presents a promising methodology for mosquito population 3 \ncontrol through HCBS methodology especially for urban and suburban areas. By providing a 4 \nsustainable and effective means of reducing mosquito populations, especially in areas endemic to 5 \ndiseases like dengue fever, yellow fever, West Nile virus, Zika, and others, we envision a 6 \nsubstantial contribution to global health initiatives. 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