The ASH1-PEX16 regulatory pathway controls peroxisome biogenesis for appressorium-mediated insect infection by a fungal pathogen.

Entomopathogenic fungi infect insects by penetrating through the cuticle into the host body. To breach the host cuticle, some fungal pathogens produce specialized infection cells called appressoria, which develop enormous turgor pressure to allow cuticle penetration. However, regulatory mechanisms underlying appressorium turgor generation are poorly understood. Here, we show that the histone lysine methyltransferase ASH1 in the insecticidal fungus Metarhizium robertsii, which is strongly induced during infection of the mosquito cuticle, regulates appressorium turgor generation and cuticle penetration by activating the peroxin gene Mrpex16 via H3K36 dimethylation. MrPEX16 is required for the biogenesis of peroxisomes that participate in lipid catabolism and further promotes the hydrolysis of triacylglycerols stored in lipid droplets to produce glycerol for turgor generation, facilitating appressorium-mediated insect infection. Together, the ASH1-PEX16 pathway plays a pivotal role in regulating peroxisome biogenesis to promote lipolysis for appressorium turgor generation, providing insights into the molecular mechanisms underlying fungal pathogenesis.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein.

BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

Effects of circadian clock disruption on gene expression and biological processes in Aedes aegypti.

BACKGROUND: This study explores the impact of disrupting the circadian clock through a Cycle gene knockout (KO) on the transcriptome of Aedes aegypti mosquitoes. The investigation aims to uncover the resulting alterations in gene expression patterns and physiological processes. RESULTS: Transcriptome analysis was conducted on Cyc knockout (AeCyc-/-) and wild-type mosquitoes at four time points in a light-dark cycle. The study identified system-driven genes that exhibit rhythmic expression independently of the core clock machinery. Cyc disruption led to altered expression of essential clock genes, affecting metabolic processes, signaling pathways, stimulus responses and immune responses. Notably, gene ontology enrichment of odorant binding proteins, indicating the clock's role in sensory perception. The absence of Cyc also impacted various regulation of metabolic and cell cycle processes was observed in all time points. CONCLUSIONS: The intricate circadian regulation in Ae. aegypti encompasses both core clock-driven and system-driven genes. The KO of Cyc gene instigated extensive gene expression changes, impacting various processes, thereby potentially affecting cellular and metabolic functions, immune responses, and sensory perception. The circadian clock's multifaceted involvement in diverse biological processes, along with its role in the mosquito's daily rhythms, forms a nexus that influences the vector's capacity to transmit diseases. These insights shed light on the circadian clock's role in shaping mosquito biology and behavior, opening new avenues for innovative disease control strategies.

CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.

Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Delayed Plasmodium falciparum Malaria in Pregnant Patient with Sickle Cell Trait 11 Years after Exposure, Oregon, USA.

Delayed Plasmodium falciparum malaria in immigrants from disease-endemic countries is rare. Such cases pose a challenge for public health because mosquitoborne transmission must be rigorously investigated. We report a case of delayed P. falciparum malaria in a pregnant woman with sickle cell trait 11 years after immigration to the United States.

Plasmodium vivax Infections among Immigrants from China Traveling to the United States.

Beginning in 2023, we observed increased Plasmodium vivax malaria cases at an institution in Los Angeles, California, USA. Most cases were among migrants from China who traveled to the United States through South and Central America. US clinicians should be aware of possible P. vivax malaria among immigrants from China.

Supreme glutathione-dependent ketosteroid isomerase in the yellow-fever transmitting mosquito Aedes aegypti.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

Detecting the impacts of humidity, rainfall, temperature, and season on chikungunya, dengue and Zika viruses in Aedes albopictus mosquitoes from selected sites in Cebu city, Philippines.

BACKGROUND: Aedes albopictus is the secondary vector for dengue virus (DENV) in the Philippines, and also harbors chikungunya (CHIKV) and Zika (ZIKV) viruses. This study aimed to determine the minimum infection rates (MIRs) of CHIKV, DENV serotypes, and ZIKV in Ae. albopictus collected from selected two-site categories by altitude (highland [H] and lowland [L] sites) in Cebu city, Philippines during the wet (WS) and dry seasons (DS) of 2021-2022, and to explore the relationships between these arboviral MIRs and the local weather. METHODS: The viral RNA extracts in pooled and reared adult Ae. albopictus collected during the DS and WS from two-site categories were subjected to RT-PCR to amplify and detect gene loci specific for CHIKV, DENV-1 to DENV-4, and ZIKV and analyzed with the weather data. RESULTS: The range of CHIKV MIRs was higher in the WS (13.61-107.38 infected individuals per 1,000 mosquitoes) than in the DS (13.22-44.12), but was similar between the two-site categories. Rainfall (RF) influenced the CHIKV MIR. The MIR ranges of both DENV-2 (WS: H = 0, L = 0; DS: H = 0-5.92; L = 0-2.6) and DENV-4 (WS: H = 0, L = 0-2.90; DS: H = 2.96-6.13, L = 0-15.63) differed by season but not between the two-site categories. Relative humidity (RH), RF, and temperature did not influence DENVs' MIRs. The MIR range of ZIKV was similar in both seasons (WS: 11.36-40.27; DS: 0-46.15) and two-site categories (H = 0-90.91, L = 0-55.56). RH and temperature influenced ZIKV MIR. CONCLUSIONS: RF influenced CHIKV MIR in Ae. albopictus, whereas RH and temperature influenced that of ZIKV. Season influenced the MIRs of CHIKV and DENVs but not in ZIKV. Ae. albopictus were co-infected with CHIKV, DENVs, and ZIKV in both highland and lowland sites in Cebu city. Recommendations include all-year-round implementation of the Philippine Department of Health's  4S enhanced strategy and installation of water pipelines in rural highlands for vector and disease control. Our findings are relevant to protect public health in the tropics in this climate change.

Tips and tools to obtain and assess mosquito viromes.

Due to their vectorial capacity, mosquitoes (Diptera: Culicidae) receive special attention from health authorities and entomologists. These cosmopolitan insects are responsible for the transmission of many viral diseases, such as dengue and yellow fever, causing huge impacts on human health and justifying the intensification of research focused on mosquito-borne diseases. In this context, the study of the virome of mosquitoes can contribute to anticipate the emergence and/or the reemergence of infectious diseases. The assessment of mosquito viromes also contributes to the surveillance of a wide variety of viruses found in these insects, allowing the early detection of pathogens with public health importance. However, the study of mosquito viromes can be challenging due to the number and complexities of steps involved in this type of research. Therefore, this article aims to describe, in a straightforward and simplified way, the steps necessary for obtention and assessment of mosquito viromes. In brief, this article explores: the capture and preservation of specimens; sampling strategies; treatment of samples before DNA/RNA extraction; extraction methodologies; enrichment and purification processes; sequencing choices; and bioinformatics analysis.

Assessing dengue risk globally using non-Markovian models.

Dengue is a vector-borne disease transmitted by Aedes mosquitoes. The worldwide spread of these mosquitoes and the increasing disease burden have emphasized the need for a spatio-temporal risk map capable of assessing dengue outbreak conditions and quantifying the outbreak risk. Given that the life cycle of Aedes mosquitoes is strongly influenced by habitat temperature, numerous studies have utilized temperature-dependent development rates of these mosquitoes to construct virus transmission and outbreak risk models. In this study, we contribute to existing research by developing a mechanistic model for the mosquito life cycle that accurately captures its non-Markovian nature. Beginning with integral equations to track the mosquito population across different life cycle stages, we demonstrate how to derive the corresponding differential equations using phase-type distributions. This approach can be further applied to similar non-Markovian processes that are currently described with less accurate Markovian models. By fitting the model to data on human dengue cases, we estimate several model parameters, allowing the development of a global spatiotemporal dengue risk map. This risk model employs temperature and precipitation data to assess the environmental suitability for dengue outbreaks in a given area.

The effect of physical barriers under a raised house on mosquito entry: an experimental study in rural Gambia.

BACKGROUND: Anopheles gambiae, the major malaria mosquito in sub-Saharan Africa, feed largely indoors at night. Raising a house off the ground with no barriers underneath reduces mosquito-house entry. This experiment tested whether walling off the space under an elevated hut affects mosquito-hut entry. METHODS: Four inhabited experimental huts, each of which could be moved up and down, were used in rural Gambia. Nightly collections of mosquitoes were made using light traps and temperature and carbon dioxide levels monitored indoors and outdoors using loggers. Each night, a reference hut was kept at ground level and three huts raised 2 m above the ground; with the space under the hut left open, walled with air-permeable walls or solid walls. Treatments were rotated every four nights using a randomized block design. The experiment was conducted for 32 nights. Primary measurements were mosquito numbers and indoor temperature in each hut. RESULTS: A total of 1,259 female Anopheles gambiae sensu lato were collected in the hut at ground level, 655 in the hut with an open ground floor, 981 in the hut with air-permeable walls underneath and 873 in the hut with solid walls underneath. Multivariate analysis, adjusting for confounders, showed that a raised hut open underneath had 53% fewer mosquitoes (95% CI 47-58%), those with air-permeable walls underneath 24% fewer (95% CI 9-36%) and huts with solid walls underneath 31% fewer (95% CI 24-37%) compared with a hut on the ground. Similar results were found for Mansonia spp. and total number of female mosquitoes, but not for Culex mosquitoes where hut entry was unaffected by height or barriers. Indoor temperature and carbon dioxide levels were similar in all huts. CONCLUSION: Raising a house 2 m from the ground reduces the entry of An. gambiae and Mansonia mosquitoes, but not Culex species. The protective effect of height is reduced if the space underneath the hut is walled off.

Evaluation of the malaria elimination programme in Muara Enim Regency: a qualitative study from Indonesia.

BACKGROUND: Malaria remains an enduring public health concern in Indonesia, exacerbated by its equatorial climate that fosters the proliferation of Anopheles mosquitoes. This study seeks to assess the performance of the malaria elimination programme comprehensively. METHODS: Between May and August 2022, a qualitative study was conducted in Muara Enim Regency, South Sumatra Province, involving 22 healthcare professionals from diverse backgrounds. These informants were strategically chosen for their pivotal roles in providing profound insights into various facets of the malaria elimination programme. This encompasses inputs such as human resources, budgetary allocation, and infrastructural support; processes like case identification and management, capacity enhancement, epidemiological surveillance, prevention measures, outbreak control, and enhanced communication and educational initiatives; and, notably, the programme's outcomes. Data were collected through 3-h Focus Group Discussions (FGDs) divided into two groups, each with 12 participants: healthcare professionals and programme managers. Additionally, in-depth interviews (IDIs) were conducted with ten informants. Employing the Input-Process-Output (IPO) model, this study meticulously analysed the healthcare system dynamics and the interventions' efficacy. RESULTS: The study unveiled many challenges during the input phase, including the absence of entomologists and a shortage of diagnostic tools. Despite these obstacles, it documented remarkable accomplishments in the output domain, marked by significant advancements in the distribution of mosquito nets and the successful implementation of the Early Warning System (EWS). Despite the adversities, the programme has made substantial strides towards malaria elimination. CONCLUSIONS: Urgent action is imperative to bolster the effectiveness of the malaria elimination programme. Key measures encompass augmenting the entomologist workforce, optimizing resource allocation, and ensuring stringent adherence to regional regulations. Addressing these concerns will enhance programme efficacy, yielding enduring public health benefits. This research substantially contributes to Indonesia's ongoing malaria elimination endeavours, furnishing actionable insights for programme enhancement. Consequently, this research holds significant importance for the malaria elimination drive.

Evaluation of a mass trapping strategy to prevent mosquito nuisance in campsites of southern France.

Mosquito traps, historically used for surveillance and research, have gained prominence as a tool for mosquito control, amidst concern over the environmental impact and increased resistance to insecticide-based methods. In this study, we tested the effectiveness of a mass trapping barrier design with two types of traps, Mosquito Magnet (MM) traps and BG-Protector (BGP) traps. This experiment was conducted in three coastal camping areas in southern France between summer and autumn 2022, where the presence of floodwater mosquito species with anthropophilic preferences like Aedes caspius represents a year-long nuisance. MM traps were set around the campsite as a barrier to interfere with mosquitoes from entering the campsites, whereas BGP traps were set within the campsites, with the aim of diverting mosquitoes away from humans at peak activity hours. Over 210,000 mosquitoes of 11 species from 4 genera were collected by both trap types across treatment campsites, with no significant differences in mosquito community samplings between BGP and MM traps. Barrier traps effectively targeted Ae. caspius, reducing total mosquito abundance in two of the three study sites by 34% and 55%. This study provides valuable insights into the efficacy and feasibility of using mass trapping barriers as a complementary control strategy for mosquito species in wetlands.

Geometric morphometrics to differentiate species and explore seasonal variation in three Mansonia species (Diptera: Culicidae) in central Thailand and their association with meteorological factors.

Mansonia mosquito species are recognised as a significant vector of human pathogens, primarily transmitting the filarial nematode, Brugia malayi. In central Thailand, the three most prevalent Mansonia species are Mansonia annulifera, Mansonia indiana and Mansonia uniformis. This study explored the influence of seasonal changes on the phenotypic variation of these Mansonia species in central Thailand using the geometric morphometrics (GM). To ensure accurate species identification, we integrated GM techniques with DNA barcoding, examining distinctions in both phenotype and genotype among the species. The intraspecific genetic divergence ranged from 0.00% to 1.69%, whereas the interspecific genetic divergence ranged from 10.52% to 16.36%. The clear distinction between intra- and interspecific distances demonstrated the presence of a barcoding gap, confirming the successful differentiation of the three Mansonia mosquito species through DNA barcoding. Similarly, the interspecies GM assessment for classifying Mansonia species demonstrated a high degree of accuracy, with an overall performance of 98.12%. Exploring seasonal variation in the three Mansonia species revealed wing variations across different seasons, and pronounced variations appearing in the cool season. Regarding their association with meteorological factors, Ma. annulifera and Ma. uniformis showed significant positive correlations with temperature (p < 0.05), and Ma. uniformis also displayed a significant negative correlation with atmospheric pressure (p < 0.05). The insights from this study will deepen our understanding of the adaptive patterns of Mansonia mosquitoes in Thailand's central region, paving the way for enhanced disease surveillance related to these vectors.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

Community perceptions, acceptability, and the durability of house screening interventions against exposure to malaria vectors in Nyimba district, Zambia.

BACKGROUND: House screening remains conspicuously absent in national malaria programs despite its recognition by the World Health Organization as a supplementary malaria vector-control intervention. This may be attributed, in part, to the knowledge gap in screen durability or longevity in local climatic conditions and community acceptance under specific cultural practices and socio-economic contexts. The objectives of this study were to assess the durability of window and door wire mesh screens a year after full house screening and to assess the acceptability of the house screening intervention to the participants involved. METHODS: This study was conducted in Nyimba district, Zambia and used both quantitative and qualitative methods of data collection and analysis. Both direct observation and questionnaires were employed to assess the durability of the screens and the main reasons for damage. Findings on damage were summarized as percentages. Focus group discussions were used to assess people's knowledge, perceptions, and acceptability of the closing eaves and house screening intervention. Deductive coding and inductive coding were used to analyse the qualitative data. RESULTS: A total of 321 out of 400 (80.3%) household owners of screened houses were interviewed. Many window screens (90.3%) were intact. In sharp contrast, most door screens were torn (n = 150; 46.7%) or entirely removed (n = 55; 17.1%). Most doors (n = 114; 76%) had their wire mesh damaged or removed on the bottom half. Goats (25.4%), rust (17.6%) and children (17.1%) were cited most as the cause of damage to door screens. The focus group discussion elicited positive experiences from the participants following the closing of eaves and screening of their windows and doors, ranging from sleeping peacefully due to reduced mosquito biting and/or nuisance and having fewer insects in the house. Participants linked house screening to reduced malaria in their households and community. CONCLUSION: This study demonstrated that in rural south-east Zambia, closing eaves and screening windows and doors was widely accepted. Participants perceived that house screening reduced human-vector contact, reduced the malaria burden and nuisance biting from other potentially disease carrying insects. However, screened doors are prone to damage, mainly by children, domestic animals, rust, and termites.