Anopheles larval ecology and physicochemical characterization of larval habitats in Dire Dawa: an area colonized by Anopheles stephensi in Eastern Ethiopia

Anopheles larval ecology and physicochemical characterization of larval habitats in Dire Dawa: an area colonized by Anopheles stephensi in Eastern Ethiopia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anopheles larval ecology and physicochemical characterization of larval habitats in Dire Dawa: an area colonized by Anopheles stephensi in Eastern Ethiopia Ephrem Abiy, Teshome Degefa, Meshesha Balkew, Hailu Merga, Denekew Zewdu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8442371/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Background : Understanding mosquito larval ecology is essential for planning and implementations of vector control strategies. The biotic and abiotic factors affect larval occurrence , density, survival and morphogenesis of mosquitoes. Artificial containers are very suitable larval habitats for some species of Anopheles and Aedes mosquitoes in urban and peri-urban settings. Therefore, this study we identified, mapped and characterized larval habitats, estimated larval density and indices larval habitats. In addition, we determined the species composition of Anopheles mosquitoes and species evenness in urban, peri-urban and rural areas of Dire Dawa city adminstration Methods : Larval habitats were surveyed and identified monthly for a period of 16 months from February 2023 to December 2024 in urban, peri-urban and rural areas of Dire Dawa. Mosquito larvae and pupae were collected and those larvae identified as Anopheles were fed on fish-food. Emerged adults were provided with 10% sucrose solution and kept under standard conditions in field insectary. Females Anopheles was identified morphologically and further species-specific PCR assay was employed to identify members of An . gambiae s.l. In addition, real time PCRassay was performed to identify An.stephensi and An.arabiensis . Water samples were taken from the larval habitats and the physico-chemical parameters were measured using HANNA Multi-parameter (H198194). Larval habitat diversity, larval abundance and distribution were assessed across the three ecological settings (urban, peri-urban and rural). Results : A total of 23, 526 larvae and 1,808 pupae of Anopheles mosquitoes were collected from 909 man-made(uncovered cemented cisterns (Brick), plastic sheets, steel drums, Tire tracks, Canal ditch, plastic tanker/Barrel) and natural habitats (River edges, ponds, animal hoof prints and swaps) in urban, peri-urban areas of Dire Dawa. The highest mean larval density (51 larvae per dip) of Anopheles mosquitoes was recorded from peri-urban sites in uncovered water tanker (brick) followed by urban site in brick (46 larvae per dip). Anopheles larvae were not found in steel drum and plastic barrels in rural sites. A total of 2,934 adult Anopheles mosquitoes were emerged from immatures collected from all sites, of which 75% (2194/2934) were An. stephensi , and 22 % (636 / 2934) were An.arabiensis . The remaining 3.0% were An. pharoensis , An. coustani , An .amharicus, and An. pretoriensis. Anopheles stephensi , An. arabiensis and An. amharicus shared the same habitats across the three ecologies. Larval density was positively correlated with availability of brick making, proximity to houses, urban setting, presence of competitors /predators, vegetation cover, shade cover and substrate type. But larval presence was not correlated with presence/absence of intervention. Larva/pupa presence were positively correlated with pH (r=0.264, p =0.01) and water pressure (r=0.21, p <0.05).There was positive correlation among temperature, electrical conductivity (EC), total dissolved solids (TDS), salinity and dissolved oxygen (DO) and negative correlation among temperature with resistivity, pH with mvPH. Larval presence was positively correlated with water salinity and pH. Conclusion: Anopheles stephensi was the predominant species found in the study area, followed by An.arabiensis, An.amharicu, An.pharoensis, An.coustani and An.pretoriensis . Uncovered water tankers (Bricks) were the most prolific artificial habitats in urban and peri-urban sites followed by plastic sheets while natural habitats such as hoof prints and river basins were the most efficient habitat types in rural and urban settings, respectively. Anopheles stephensi was found in natural habitats of Butuji and legehare rivers from urban site, and in rural sites from man-made habitat of plastic sheet. Anopheles amharicus larvae was found in plastic sheet, an artificial habitat common in urban, peri-urban and rural areas. We report here the occurrence of An.stephensi in rural areas, breeding in natural habitats, and co-existing with An. gambiae s.l complex. Availability of brick making, shorter distance from living houses being in urban ecology were directly correlated with larval density. Habitat abundance and positivity of uncovered water tankers (cemented cisterns or bricks) in urban and pre-urban sites could indicate for feasibility and proper implementation of larval source management. Physico-chemical characterization mosquito larval habitat Anopheles stephensi larval density Ethiopia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Anopheles larval habitat characterization refers to the process of identifying and describing the environmental conditions where Anopheles mosquitoes lay their eggs and where their larvae develop [ 1 ]. These habitats are typically shallow bodies of water, such as ponds, swamps, streams, rice fields, and containers, which can vary in size and water chemistry. The characterization includes factors like water depth, vegetation, temperature, pH, salinity, and the presence of organic matter, as these conditions influence the survival and development of the larvae [ 2 – 4 ]. Understanding these habitats is crucial for malaria control, as targeting larval habitats with appropriate interventions, such as biological or chemical control methods, can help reduce mosquito populations and limit the transmission of malaria. Each malaria vector has its own larval habitat preferences for oviposition that might be linked with their genetics make up or environmental parameters that imposed behavioral variation [ 5 ]. The main malaria vector An.gambiae complex prefers to breed in clean water of temporary rain pools, hoof prints, natural pockets of river, saline water (some species).The An.funestus groups breed in emergent vegetation and year round river sides, breeds in rice fields, and irrigation schemes. But the invasive An.stephensi breeds in artificial small containers, jars, bricks, plastic sheets, barrels, iron tankers, steel drums, used tire, ditches sometimes even in polluted water and fountains [ 6 , 7 ]. Mosquito breeding habitats have specific biotic and abiotic factors that make them suitable to oviposition and support development of mosquito life cycle or metamorphosis [ 1 , 8 ]. Studies conducted in Ethiopia on Anopheles gambiae s.l. showed that the presence of predators, presence of other competing mosquito species, vegetation coverage and intervention, water temperature, electrical conductivity, pH, dissolved oxygen, salinity, total dissolved solution and turbidity are among the physicochemical factors that affect the density and distribution of mosquito larvae [ 9 , 10 ]. Similar studies in India, Benin and Iran indicated the correlation between environmental factors and Anopheles stephensi and other Anopheles spss larval abundance [ 11 – 13 ].But there are no studies that demonstrated the correlation between water physicochemical parameters and Anopheles stephensi larval abundance in Ethiopia. Globally, LSM (larval source management) has a proven track record in malaria control and even elimination in some countries. Historical successes in Brazil, Egypt, and parts of Asia relied heavily on environmental management [ 14 ]. More recent studies show that larviciding using biological agents like Bacillus thuringiensis israelensis (Bti) can reduce larval and adult vector densities by up to 90% [ 15 ].In Ethiopia, larval source management is assumed to be a supplementary strategy besides the principal vector control interventions : use of insecticide treated nets and indoor residual spraying but there is inadequate larval identification and response to guide adoption of LSM approaches [ 16 ]. In Ethiopia, pilot studies have shown promising results with significant reductions in larval density and adult vector population reduction through regular Bti and Sumilarv application [ 17 , 18 ] .Similarly, a study in Eastern part of the country on larval habitat suggested that LSM could be effective for focused control of An.stephensi [ 19 ]. In addition, LSM integrated with LLINs and IRS resulted in a notable reduction in malaria incidence in Tolay area of oromia region and in northern Cot^e Idvore’ ([ 20 , 21 ]. In contrary, Ayana et al, 2025 reported that there was an increased malaria cases irrespective of LSM implementation and need to investigate factors that might affect the success of the intervention [ 22 ]. Therefore, this study was conducted to assess the physicochemical characteristics of larval habitats and relation with the ecology, season, larval density and distribution of larvae across the urban, peri-urban and rural areas of Dire Dawa. Methods Study area The study was conducted in three ecological settings, of Dire Dawa City administration (515 Km East of Addis Ababa) which included urban, per-urban and rural settings. The urban setting considered for this study were Dire Dawa University, GTZ and Mermersa. The peri-urban sites were those around the industrial zone including villages of Melka-Jebdu and Jerba. The rural study sites were Aseliso, Hulahulul and Boren. The rural sites were agricultural sites with grass land and scanty vegetation coverage. (Fig. 1 )[ 23 ]. During the study, larval source management was implemented jointly by the President’s Malaria Initiative (PMI) and the Ministry of Health of Ethiopia in the urban and peri-urban sites [ 24 ]. The study area has seasonal malaria transmission, with Plasmodium falciparum and P. vivax the main parasite species,and the vectors include Anopheles arabiensis and An. phareonsis. Recent studies reported the occurrence of An. stephensi as an additional vector [ 25 , 26 ]. Immature mosquito collections and larval habitat characterization Larval surveys were conducted following standard WHO protocol and mosquito immatures were collected using larval dipper of 350ml capacity [ 27 , 28 ]. Twenty dips were taken from each habitat by dipping at different sides of the habitat. Each larval instar and pupae were counted and recorded. Data were entered monthly using open data kit (ODK) immediately synced to the International Centers of Excellence for Malaria Research (ICEMR) database server to avoid data loss. Geographic coordinates, ecological setting (urban, peri-urban and rural), habitat type (Artificial and Natural) and picture of the habitat on spot, source of water (tap ,rain, or river), distance from household, land use (surrounding environment of the habitat), shade status/degree of exposure to sunlight, water flow, substrate type, distance from brick making, distance from nearest resting habitat and frequency of utilization, presence of canopy cover, presence of intervention, type of intervention and presence of predators were included among others. Mosquito immatures were classified as Anopheline, Aedine and Culicine based on morphological observations. The species composition of Anopheles mosquitoes were determined from adults reared from larvae or pupae. Molecular species identification The collected adult Anopheles were identified first to species using the morphological keys of Coetzee et al . 2020 and Glich et al . 1995 [ 29 , 30 ]. Those identified as An. gamabiae s.l were further re-identified using species specific PCR. Real time PCR was also employed to confirm An. stephensi at the Genomics Laboratory of Jimma University Tropical Infectious and Diseases research center (TIDRC). DNA extraction and amplification DNA extraction was done using both the Chelex and Extracta protocols [ 31 ]. PCR amplification was carried out according to the methods of Scott et al . [ 32 ] using species-specific primers for An . arabiensis (AR: 5′-AAG TGT CCT TCT CCA TCC TA-3′) and An . amharicus , formerly Anopheles quadriannulatus B (QD: 5′-CAG ACC AAG AGA GAT GGT TAG TAT-3′)[ 33 ]. Anopheles gambiae (GA: 5′-CTG GTT TGG TCGGCA CGT TT-3′) and a universal primer (UN: 5′-GTGTGC CCC TTC CTC GATGT-3′). Then the amplicon was loaded on a 2% agarose gel stained with ethidium bromide and run for gel electrophoresis. Anopheles arabiensis from the Sekoru insectary colony and An . amharicus specimens from Arjo area were used as positive controls [ 34 ]. Physico-chemical analysis of larval habitats Physicochemical parameters of each habitat were measured using HANNA (H198184) instrument of multi-parameters (Multiparameter Meter, Hanna Instruments Inc., Woonsocket, Rhode Island, 02895, USA) after proper calibration of the instrument with the manufacturer’s probe solutions that was provided together with multi-parameter. Measurements of acidity and alkalinity (pH/mV), oxidative reduction potential (ORP), electrical conductivity (EC), total dissolved solids (TDS), resistivity, salinity, seawater, dissolved oxygen (DO), atmospheric pressure and temperature were taken from 102 larval habitats. Statistical Analysis Data were entered in to SPSS version 27 and descriptive analysis was used to compare frequency, distribution, and magnitude of Anopheles larvae in the urban, peri-urban and rural interfaces. Multiple regression analysis was used to compare the effect of physicochemical variables of habitats on the Anopheles larva presence / absence, and larval density. Principal component analysis (PCA) was performed and Spearman’s correlation coefficient were used to dictate the correlation among water physicochemical parameters and larval presence and density. Cross tabs, Pearson’s Chi squared and Fisher’s exact tests were used to compare the association of presence/absence of predators, and presence/absence of intervention on larval presence and density. Larval density was calculated as number of larvae/pupae per 20 dips per habitat. Habitat index, container Index, and breteau Index were used to compare the larval density and abundance in each of the three ecological settings. Standard mathematical formulae [ 35 ] were used to calculate the larval indices as follows: 1) House Index (HI) was calculated by dividing the number of houses infested to the total number of houses inspected and multiplied by 100. 2) Container Index (CI) was calculated by dividing the number of containers positive for anopheles larvae to the total number of containers inspected and multiplied by 100. 3)Breteau Index (BI) was calculated by dividing the number of positive containers to total number of houses and multiplied by 100. Results Larval habitat abundance and indices A total of 909 habitats were surveyed in Dire Dawa, of which, 857 (94.3%) were artificial breeding habitats such as bricks/concrete water tankers, plastic sheets, plastic water tankers (barrels), steel drums, canal ditches and tire tracks while the other were natural habitats such as river edges, animal hoof prints, ponds and swamps (Table 1 )( Fig. 2 ). In Dire Dawa, uncovered water tanker/Cemented cistern or brick were the predominant habitat type 520/909 (57.2%) and followed by plastic sheet 221/909 (24.3%). Anopheles larvae were found in 297 (32.7%) of the houses surveyed and in 400(43.7%) of containers inspected in Dire Dawa (Table 1 and Table 2 ). Table 1 Larval habitat abundance, proportion and larval presence positivity in urban, peri-urban and rural sites in Dire Dawa City Administration (2024) Habitat type n*(%) positive for Anopheles(%) Brick concrete water tanker 520 (57.2) 187(36.0) Drainage ditch/canal 10 (1.1) 2(20.0) River edge 25 (2.8) 14 (56.0) Tire Tracks 6 (0.7) 4 (66.6) Foot Print 11(1.2) 9 (81.8) Swamp 6 (0.6) 4 (66.7) Pond 10 (1.2) 4 (40.0) Steel Drum 25 (2.8) 5 (20.0) covered drinking water tanker/Birca 60 (6.6) 8 (13.3) Plastic sheet 221(24.3) 58 (26.2) Other (small water containers, pipe, foundation, septic tank) 15 (1.7) 2 (13.3) Total 909 (100) 297 (33) As depicted below in Table 2 , about 42, 34%, and 24% of larva habitats surveyed were from urban, peri-urban and rural settings respectively. Nine hundred-fifteen containers were found in houses surveyed with high number of containers 350(38.3%) in urban, 316 (34.5%) in peri-urban and 249 (27.2%) in rural sites respectively. Table 2 Larval indices and the distribution of habitats in urban, peri-urban and rural ecologies in Dire Dawa City Administration, Ethiopia Site Houses surveyed n (%) # positive houses n (%)HI* #containers n (%) #Positive containers CI* n (%) Breteau Index BI* n (%) Urban 385 (42.0) 133 (34.5) 350 (38.3) 175 (50) 175 (45.5) Peri-urban 307 (34.0) 107 (34.8) 316 (34.5) 130 (41.1) 130 (42.3) Rural 217 (24.0) 57 (26.3) 249 (27.2) 95 (38.2) 95 (43.8) Total 909 (100) 297 (32.7) 915 (100) 400 (43.7) 400 (44) N = number, *HI-House index, CI:Container index and BI: Breteau index A total of 23,526 larvae and 1,808 pupae of Anopheles mosquitoes were collected from 909 habitats in urban, peri-uban, and rural areas of Dire Dawa. There was a difference in monthly larval density at each ecology and habitat type. Paired-samples t-tests indicated that there is a significant difference in the density of Anopheles larvae across months ( t=-3.27, n = 34, p = .003) with a mean difference of − 6.98 larvae per dip (95% CI: −11.32 to − 2.63) and a medium effect size (Cohen’s dₚ = 0.55), indicating a moderate seasonal influence on larval abundance. Similarly, the paired-samples t-test showed that larval density differed significantly between ecological categories ( t = − 5.37, n = 34, p < .001) with a mean difference of − 11.52 larvae per dip (95% CI: −15.88 to − 7.16) and a large effect size (Cohen’s dₚ = 0.91). A stronger effect was observed across ecological settings, which suggests that ecological characteristics exert a greater influence on Anopheles larval density than seasonal variation alone. In urban sites, where larval density was estimated to be constant throughout the year, the monthly Anopheles larval density was high in October and lowest in May in urban settings. In peri-urban sites, Anopheles larval density was highest in October and absent in May. In rural sites, larval density was peak in February and absent from June to October (Fig. 3 ), showing that there was a significance difference between months and ecology, where some ecological settings were absent in some months ( t = 7.38, n = 34, p < 0.001), suggesting that temporal and ecological factors together influence larval dynamics. The mean larval density were highest in peri-urban site in artificial uncovered water tanker (51 larvae per dip), followed by natural ditch in urban site (46 larvae per dips), and lowest in rural and urban plastic sheet habitats, and lowest in plastic sheets in Urban sites (0 per dip/habitat). Habitat type differs significantly across the season (t = 6.36, n = 77, p < 0.001) indicating seasonal variation affect habitat type. Anopheles larvae abundance and density were significantly different in different types of habitats (t = -4.8, n = 77, p < 0.001). There is a significant association between ecological settings (urban, peri-urban, and rural) and habitat types (t=-6.027, n = 77, p < 0.001). Seasonal variations, specifically dry and long rain seasons, strongly affect larval abundance and density (t = -4.95, n = 77, p < 0.001). As construction of houses and production of bricks are common in urban and peri-urban areas of Dire Dawa, Anopheles larvae population density was high throughout the year in the artificial water storages, regardless of the seasonal variation in the urban and peri-urban ecologies. But in rural areas, Anopheles larval density increased following the short rain season (Fig. 4 ). Proximity of larval habitats to houses 80% (732 /909) were located in less than 50 meters from nearby living houses, 65% (589/909) of the habitats were at 0–50 meters away from brick making houses. Additionally, 56% (509/909) of the habitats surveyed were surrounded by construction site or land-use for brick making. Half of the total habitats substrate were made of concrete with 35% (320/909) covered with plastic sheet or lining and about 11% of the habitat substrate was mud. The results of logistic regression analysis indicated that man-made/artificial habitat and grass land of surrounding were significant predictors for the presence of Anopheles larvae/pupae (Table 3 ). Table 3 Logistic regression analysis for the assessment of predictors of mosquito larval occurrence in urban, peri-urban and rural areas of Dire Dawa City Administration, Ethiopia Variable Estimate ChiSquare Prob > ChiSq Lower 95% Upper 95% Eco setting[peri-urban] 0.249 1.34 0.2469 -0.172 0.675 Eco setting[rural] -0.251 1.03 0.3103 -0.747 0.228 Habitat category[man made] 2.222 5.76 0.0164 0.823 4.805 Landuse2[construction] 0.199 0.19 0.6608 -0.638 1.148 Landuse2[farmland] 0.407 0.7 0.4023 -0.534 1.386 Landuse2[grassland] 1.504 4.45 0.0349 0.165 3.019 Landuse2[house] -0.588 1.05 0.3058 -1.717 0.557 Landuse2[road] -1.943 1.28 0.2587 -5.645 1.139 Vegetation Cover -0.008 0.23 0.6321 -0.041 0.027 Shade coverage 0.001 0.01 0.942 -0.018 0.018 LSM -0.525 0.88 0.3494 -1.693 0.540 Distance to house -0.004 2.26 0.1325 -0.010 0.001 Distance to resting 0.000 0 0.988 -0.005 0.005 Distance to brick making -0.003 1.69 0.1932 -0.009 0.001 Habitat length -0.031 0.86 0.3531 -0.107 0.032 Both natural and man-made habitats were having same correlation with Anopheles and Culex larval presence and Aedes larval density. But rural sites were highly correlated with Aedes larval density than urban and peri-urban and sites. Conversely, An.stephensi larval presence were correlated with urban and peri-urban than rural sites. Similarly, the result of the PCA indicated positive correlation between land use (surrounding environment) and larval presence, habitat with in Grass-land environment, farm land, and availability of shrubs and in or near household were correlated with presence of Anopheles, Culicines and Aedeines larvae (Fig. 5 and Fig. 7 ). The results of principal component analysis (PCA) indicated that Larva/pupa presence were positively correlated with pH (r = 0.264, p = 0.01) and water pressure (r = 0.21, p < 0.05). Similarly, there was a significant positive correlation with temperature and conductivity (EC) (r = 0.31, p = 0.02), Total dissolved solids (TDS) ((, r = 0.38, p = 0.02) and, Salinity (r = 0.38, p = 0.02)(Fig. 6 ). EC, TDS and salinity were positively correlated (r = 1, p = 0.00). Also Dissolved oxygen (DO) and water pressure (psi) showed positive correlation (r = 0.211, p = 0.040). But, temperature and resistivity had negative correlation (r = − 0.494, p = 0.00), pH and mvpH also had negative correlation (r= -0.993, p = 0.00). In addition, there was no significant correlation between temperature and Dissolved oxygen, Dissolved oxygen and Salinity ( p > 0.05). Oxydative reduction potential (ORP) was the variable that had no-significant correlation with the dependent variable (Fig. 6 ). The PCA result also showed that Anopheles larval/pupa density were positively correlated with shorter distance to living house and distance to resting places, availability of brick making, vegetation cover, shading, pH and salinity (Fig. 7 ). The Pearson’s Chi squared test revealed a significance association between the predators and competitors presence on larval presence (ꭓ 2 =16.3, df:1, p < 0.01). Larvae were found more often in locations where predators were present (66.7%) compared to locations without predators (31.5%).The result indicated that larva presence and predator presence are not independent, and predator and competitors presence is significantly associated with higher likelihood of larva presence. But, the Pearson’s chi squared test indicated that there is no significance association between presence of intervention (larviciding) and larval presence (ꭓ 2 =2.66, df = 2, p = 0.264), the presence of intervention doesn’t affect larval presence ( p > 0.05). As depicted in Fig. 5 , Clear water had positive correlation with presence of Anopheles stephensi. Moreover, water salinity, water temperature and water pH were among the strong predictors of the presence of An.stephensi larvae (Fig. 7 ) The analysis of water salinity revealed that the majority of samples (88.3%) fell within the valid or normal salinity range, indicating predominantly fresh water conditions across the study area. Only 11.7% of the total 873 samples exhibited measurable salinity above 0 ppt. Among these, slightly saline water (2–3 ppt) was the most frequent, accounting for 5.6% of the samples, followed by low salinity levels between 0–1 ppt (2.4%). Higher salinity categories were much less common, with only 1.8% of samples in the 4–5 ppt range and less than 1% exceeding 6 ppt. Extremely saline samples (> 30 ppt) represented just 0.3% of the total (Table 5 ). Table 4 Results of water salinity measurements of larval habitats in Dire Dawa City Administration, Ethiopia Salinity (ppt) N (%) Category 0–1 21(2.4) Very low salinity (freshwater). 2–3 49 (5.6) Slightly saline (brackish tendency). 4–5 16 (18) Moderately saline. 6–7 6 (0.7) Noticeably saline. 8–9 2 (0.2) Highly saline. 10–14 5 (0.6) Very saline—approaching seawater levels. > 30 3 (0.3) Extremely saline (seawater or hyper-saline). The water physicochemical analysis indicated that water temperature of larval habitat ranged from 19°C to 34°C. The water temperature for 80.4% (82/102) of larval habitats was from 20–27°C (room temperature), 16.6% (17/102) of the larval habitats water temperature was from 28–34°C, and for the remaining 2.9% (3/102) of habitats, the water temperature recorded were 19°C. The water pH values measured and categorized in to three as acidic water (pH value 7).The result indicated that 86.3% (88/102) of habitats were alkaline, 12.7% (13/102) were neutral and 0.98% (1/102) of habitats were acidic. Five types of substrate were considered (Gravel, Sand, Mud, Concrete and Plastic cloth) and the result of the study showed that 50% (52/102) of the habitats substrate were made of concrete and 25% (26/102) of the habitat substrate were made of plastic cloths/container. Fisher’s exact test indicated that there is a statistical association between larval presence and substrate type (ꭓ 2 =26.6, df = 6, p < 0.01). Species diversity and evenness A total of 2,934 adult Anopheles mosquitoes were emerged from immatures from all sites and An. stephensi was the predominant species 75% (2194/2934) followed by An.arabiensis 22% (636 / 2934), An. pharoensis 1.7% (50/2934), An. coustani 1.3% (38/2934), An.amharicus 0.3% (9/2934) and An. pretoriensis 0.2% (7/2934), respectively (Table 5 ). Table 5 Anopheles mosquito species diversity, richness and evenness by habitat type in Dire Dawa City Administration, Ethiopia Habitat Species Abundance Proportion (pi) Lnpi pi*Lnpi Shannon diversity index (H') Species eveness (J): H/LnS Species richness (S) Artificial An.stephensi 1873 0.91 -0.1 -1. 0.1 0.3 3 An.arabiensis 185 0.09 -2.4 -0.2 0.22 An.amharicus 8 0.00 -5.6 -0.1 0.02 An.pretoriensis 0 0 0 0 0 An coustani 0 0 0 0 0 An.pharoensis 0 0 0 0 0 Total 2066 1 0 0 0.33 Natural An.stephensi 321 0.37 -1 -0.4 0.4 0.6 6 An.arabiensis 451 0.52 -1 -0.3 0.3 An.amharicus 1 0.00 -7 -0.01 0.01 An.pretoriensis 7 0.01 -5 -0.04 0.04 An.coustani 38 0.04 -3 -0.14 0.14 An.pharoensis 50 0.06 -3 -0.16 0.16 Total 868 1 0 0 1.06 Further paired samples t -test revealed significant differences in species composition between habitat categories (man-made and natural). Abundance differed significantly between habitats ( t = − 2.22, n = 13, p = 0.004), with man-made habitats having higher Anopheles abundance. Significant habitat-related differences were also observed in Shannon diversity (t = 10.03, n = 13, p < 0.001), species evenness (t = 5.70, n = 13, p < 0.001), and species richness (t = 5.7, n = 13, p < 0.001), indicating greater diversity, more even species distribution, and higher species richness in natural habitat category. An. stephensi , An.arabiensis and An.amharicus found to share same artificial habitats more specifically plastic sheets (in urban, peri-urban and rural), and in natural habitats more specifically a river Butuji river found in Dire Dawa city. Molecular identification of An.gambiae s.l and An.stephensi Of total 645 An.gambiae s.l sub-samples that were tested using PCR, 98.6% (n = 636) were found to be An.arabiensis , and 1.4% (n = 9) were An.amharicus . Similarly, real time PCR (qPCR) was performed to confirm morphologically identified An.stephensi specimens from An.gambiae s.l, and accordingly 200 sub-samples of each An.stephensi and An.arabiensis were proved distinct. Discussion Our study indicated that An.stephensi immature stages are abundant throughout the year regardless of the seasonal variation of rain, with peak larval densities in dry season in all the three ecological settings. Similar results were reported by Yared et al . 2023 [ 19 ] in Somali region, Jigjiga town, where larvae were present in dry season. This might be because both Dire Dawa city and Jigjiga town have similar in expansion of urbanization and construction which support the breeding of immature stages in dry season. Moreover, larval habitats were found in less than 50m from living houses and brick making houses were located at distance ranging from 0-50m away from living houses. This finding has an important implication as far as supporting the life cycle of Anopheles and maximizing the probability of man vector contact. This finding could help to implement targeted larviciding, larval source management in habitats that harbor the immature stages throughout the year. The other finding of our study is the co-existence of An.stephensi with An.gambiae s.l , sharing same both artificial and natural habitats and ecological settings. An.stephensi were known to be vector of malaria mainly in urban settings and in an artificial habitats [ 6 , 11 ], but in our study An.stephensi were found in rural ecology in Dire Dawa, and in river edges of Legehare and Butuji rivers, the two known and permanent habitats in the city. This further remarks the expansion of An.stephensi to rural sites and adapting natural habitats to breed. Our finding coincides with a study done in South east Iran where An.stephensi were found in river edges with sandy substrates [ 12 ]. Unexpected result of our research in the study area is, the presence of An.amharicus which were reported in South Western, Central, Western, [ 33 , 36 ] and most recently in North west part of the country, here in the Eastern part of Ethiopia ,we report the existence of An.amharicus in urban,peri-urban and rural ecologies of Dire Dawa for first time. Additionally, An.coustani , An.pretoriensis and An.phareonsis were found in the study area. This finding recommends further investigation on the role of An.amharicus and the other vectors in malaria transmission in Dire Dawa and their general bionomics including its susceptibility to current insecticides in use are important. Water physicochemical analysis indicted that larval distribution and abundance was not significantly different as the measures of the water physicochemical vary. Also a change in Ecology, season, habitat category doesn’t indicate a difference on presence and absence of larvae and density of larvae in man-made habitats. Our study align with the study conducted in Northern Iran which indicated no significance difference in physicochemical characteristics and larval density [ 37 ]. In contrast to our study, that indicated the analysis of water salinity revealed that the majority of samples (88.3%) fell within the valid or normal salinity range and the 86% fell in alkaline pH, indicating predominantly fresh water conditions across the study area, studies on correlation of Anopheles larval density with physicochemical characteristics in Benin and South east Iran indicated that there was high positive correlation of larval density with temperature [ 13 ], dissolved oxygen and salinity [ 12 , 13 ].This difference might be because of the difference in the source of water in urban areas of Benin and South east Iran from our study area where almost 98% of source of water for larval habitats were ground water and chemical characters were in similar ranges. In agreement with a study conducted by Mereta et al. 2013 [ 9 ], our study found that there was wide spread distribution of Anopheles larvae in small man-made aquatic habitats but in contrast to Mereta eta al. 2013 finding, in our study, Anopheles larva was abundant in both natural and artificial habitats. Year- round presence of larvae in artificial habitats indicated larval density, distribution and abundance were not affected by season as far as water supply continued for the purpose of mainly for construction and brick making. Expansion of An.stephensi to rural sites could be due to the rapid urbanization and industrial expansion which is accompanied with construction of water storages. The An.stephensi samples found in rural sites (40km away from Dire Dawa city) in Aseliso was in plastic sheet habitat type implicated that where ever the artificial habitats are used they are suitable for the exotic species to breed in. This study indicated the strong association of some ecological settings with the density and abundance of An.stephensi in urban and peri-urban than rural sites this could be due to availability of enormous construction and brick manufacturing in urban and peri-urban settings. Conclusion An.stephensi was the predominant species found in the study area, followed by An.arabiensis, An.amharicu, An.pharoensis, An.coustani and An.pretoriensis . Uncovered water tankers were the most efficient habitat types in urban and peri-urban sites followed by plastic sheet habitat types while hoof prints and river basins under natural habitats were the most efficient habitat types in rural and urban sites respectively. An. stephensi was found in rivers of urban site and plastic sheets of rural sites co-existing with An.arabiensis in same sites. Both shared same ecological settings and habitat types. An.amharicus was found to breed in artificial habitats of the three ecological settings. We report here the existence of An.amahricus for the first time in the study area, Eastern part of Ethiopia, expansion of An.stephensi in rural areas, breeding in natural habitats, and co-existing with An gambiae s.l . Availability of brick making, shorter distance from living houses being in urban ecology were directly correlated with larval density. The result of our study revealed that there is significant correlation of ecological and seasonal variation in the density of Anopheles larvae. Habitat abundance and positivity of uncovered water tankers in urban and pre-urban sites could indicate for feasibility and proper implementation of larval source management. Limitation of the study The physicochemical tests were performed in a cross-sectional way but we believe to be done during main rain season to observe if seasonal variation affects the water chemistry. Declarations Funding This study obtained financial support from the National Institutes of Health (D43 TW001505, R01 A1050243 and U19 AI129326). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Authors’ contributions EA, MB, GY and DY conceived and designed the study; EA, HM, HA and TD were involved in data collection and field supervision. EA, TD and AA performed laboratory analysis. EA, ML, DZ and GZ performed the data analysis and EA drafted the manuscript. ML developed map of the study area and figures. TD, MB, DY and GY critically reviewed the manuscript. All the authors read and approved the final manuscript. Ethics approval and consent to participate Ethical clearance and amendment was obtained from the institutional review board (IRB) of the Institute of Health, Jimma University (Reference number) JUIH/IRB/236/25. Furthermore, permission was obtained from the Dire Dawa Health Bureau and health facilities. Consent for publication Not applicable Competing interest The authors declare that they have no competing interests References WHO. Manual on practical entomology in Malaria. Part II. Geneva: World Health Organization; 1975. Wilson ML, Krogstad DJ, Arinaitwe E, Arevalo-Herrera M, Chery L, Ferreira MU, Ndiaye D, Mathanga DP, Eapen A. Urban malaria: understanding its epidemiology, ecology, and transmission across seven diverse ICEMR network sites. Am J Trop Med Hyg. 2015;93:110. Silver JB. Mosquito ecology: field sampling methods. springer science & business media; 2007. Githeko A, Mbogo C, Atieli F. Resting behaviour, ecology and genetics of malaria vectors in large scale agricultural areas of Western Kenya. Parassitologia. 1996;38:481–9. Jaenike J. Genetics of oviposition-site preference in Drosophila tripunctata. Heredity. 1987;59:363–9. Taylor R, Messenger LA, Abeku TA, Clarke SE, Yadav RS, Lines J. Invasive Anopheles stephensi in Africa: insights from Asia. Trends Parasitol. 2024;40:731–43. Balkew M, Mumba P, Yohannes G, Abiy E, Getachew D, Yared S, Worku A, Gebresilassie A, Tadesse FG, Gadisa E. An update on the distribution, bionomics, and insecticide susceptibility of Anopheles stephensi in Ethiopia, 2018–2020. Malar J. 2021;20:263. Service M. Medical entomology for students. 2000. Mereta ST, Yewhalaw D, Boets P, Ahmed A, Duchateau L, Speybroeck N, Vanwambeke SO, Legesse W, De Meester L, Goethals PL. Physico-chemical and biological characterization of anopheline mosquito larval habitats (Diptera: Culicidae): implications for malaria control. Parasit Vectors. 2013;6:320. Tsegaye A, Demissew A, Hawaria D, Abossie A, Getachew H, Habtamu K, Degefa T, Wang X, Lee M-C, Zhou G. Anopheles larval habitats seasonality and environmental factors affecting larval abundance and distribution in Arjo-Didessa sugar cane plantation, Ethiopia. Malar J. 2023;22:350. Thomas S, Ravishankaran S, Justin JA, Asokan A, Mathai MT, Valecha N, Thomas MB, Eapen A. Overhead tank is the potential breeding habitat of Anopheles stephensi in an urban transmission setting of Chennai, India. Malar J. 2016;15:274. Soleimani-Ahmadi M, Vatandoost H, Zare M. Characterization of larval habitats for anopheline mosquitoes in a malarious area under elimination program in the southeast of Iran. Asian Pac J Trop Biomed. 2014;4:S73–80. Hessou-Djossou D, Djègbè I, Ahadji-Dabla KM, Nonfodji OM, Tchigossou G, Djouaka R, Cornelie S, Djogbenou L, Akogbeto M, Chandre F. Diversity of larval habitats of Anopheles mosquitoes in urban areas of Benin and influence of their physicochemical and bacteriological characteristics on larval density. Parasit vectors. 2022;15:207. Keiser J, Singer BH, Utzinger J. Reducing the burden of malaria in different eco-epidemiological settings with environmental management: a systematic review. Lancet Infect Dis. 2005;5:695–708. Fillinger U, Kannady K, William G, Vanek MJ, Dongus S, Nyika D, Geissbühler Y, Chaki PP, Govella NJ, Mathenge EM. A tool box for operational mosquito larval control: preliminary results and early lessons from the Urban Malaria Control Programme in Dar es Salaam, Tanzania. Malar J. 2008;7:20. Ethiopia FMoH. Ethiopia malaria elimination strategic plan: 2021–2025. 2020. Teshome A, Erko B, Golassa L, Yohannes G, Irish SR, Zohdy S, Dugassa S. Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in Ethiopia. Malar J. 2023;22:48. Yewhalaw D, Erena E, Degefa T, Kifle YG, Zemene E, Simma EA. Biological and residual activity of candidate larvicide formulation, SumiLarv 2MR, against an exotic invasive mosquito Anopheles stephensi Liston, 1901 (Diptera: Culicidae) in Ethiopia. Scie Rep. 2025;15:291. Yared S, Gebresilassie A, Aklilu E, Abdulahi E, Kirstein OD, Gonzalez-Olvera G, Che-Mendoza A, Bibiano-Marin W, Waymire E, Lines J. Building the vector in: construction practices and the invasion and persistence of Anopheles stephensi in Jigjiga, Ethiopia. Lancet Planet Health. 2023;7:e999–1005. Mutero CM, Okoyo C, Girma M, Mwangangi J, Kibe L, Ng’ang’a P, Kussa D, Diiro G, Affognon H, Mbogo CM. Evaluating the impact of larviciding with Bti and community education and mobilization as supplementary integrated vector management interventions for malaria control in Kenya and Ethiopia. Malar J. 2020;19:390. Tia J-PB, Tchicaya ES, Zahouli JZ, Ouattara AF, Vavassori L, Assamoi J-B, Small G, Koudou BG. Combined use of long-lasting insecticidal nets and Bacillus thuringiensis israelensis larviciding, a promising integrated approach against malaria transmission in northern Côte d'Ivoire. Malar J. 2024;23:168. Ayana GM, Jalilian A, Ashine T, Molla E, Hailemeskel E, Yemane DH, Yirgu H, Negash N, Teferi N, Teshome D. Larval source management in Ethiopia: modelling to assess its effectiveness in curbing malaria surge in dire Dawa and Batu Towns. Malar J. 2024;23:366. Esri. ArcGIS Pro Version 3.5 [Computer software].Redlands,CA:Esri. (2025). 2025. 2023. TPVP: Ethiopia Final Entomological Report, April 2022-March 2023. 2023. Emiru T, Getachew D, Murphy M, Sedda L, Ejigu LA, Bulto MG, Byrne I, Demisse M, Abdo M, Chali W. Evidence for a role of Anopheles stephensi in the spread of drug-and diagnosis-resistant malaria in Africa. Nat Med. 2023;29:3203–11. Merga H, Degefa T, Birhanu Z, Abiy E, Lee M-C, Yan G, Yewhalaw D. Urban malaria and its determinants in Eastern Ethiopia: the role of Anopheles stephensi and urbanization. Malar J. 2024;23:303. WHO. Manual on practical entomology in Malaria. Part II. Methods and techniques. 1975. Organization WH. Malaria entomology and vector control. In Malaria entomology and vector control. 2013. Coetzee M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). Malar J. 2020;19:1–20. Glick JI. Illustrated key to the female Anopheles of southwestern Asia and Egypt (Diptera: Culicidae). Mosq Syst. 1992;24:125–53. Musapa M, Kumwenda T, Mkulama M, Chishimba S, Norris DE, Thuma PE, Mharakurwa S. A simple Chelex protocol for DNA extraction from Anopheles spp. J Vis Exp 2013:3281. Scott JA, Brogdon WG, Collins FH. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993;49:520–9. Coetzee M, Hunt RH, Wilkerson R, Della Torre A, Coulibaly MB, Besansky NJ. Anopheles coluzzii and Anopheles amharicus , new members of the Anopheles gambiae complex. Zootaxa. 2013;3619:246–74. Demissew A, Hawaria D, Kibret S, Animut A, Tsegaye A, Lee M-C, Yan G, Yewhalaw D. Impact of sugarcane irrigation on malaria vector Anopheles mosquito fauna, abundance and seasonality in Arjo-Didessa, Ethiopia. Malar J. 2020;19:344. Silver JB. Mosquito ecology: field sampling methods. Springer; 2008. Demissew A, Animut A, Kibret S, Tsegaye A, Hawaria D, Degefa T, Getachew H, Lee M-C, Yan G, Yewhalaw D. Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia. PLoS ONE. 2022;17:e0261713. Nikookar SH, Fazeli-Dinan M, Azari-Hamidian S, Mousavinasab SN, Aarabi M, Ziapour SP, Esfandyari Y, Enayati A. Correlation between mosquito larval density and their habitat physicochemical characteristics in Mazandaran Province, northern Iran. PLoS Negl Trop Dis. 2017;11:e0005835. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8442371","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578414396,"identity":"15ad7a2c-7201-426f-97f0-5cf01f7411d0","order_by":0,"name":"Ephrem 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1","display":"","copyAsset":false,"role":"figure","size":701544,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the study area in Dire Dawa City Administration, 2024.This map was created with ArcGIS Pro 3.5 by Esri.(www.esri.com).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/e151a34b7e3271e8a88bd371.png"},{"id":100938368,"identity":"f028b6a3-ac2d-4202-b3ac-112324db7778","added_by":"auto","created_at":"2026-01-23 03:45:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":899689,"visible":true,"origin":"","legend":"\u003cp\u003eTypes of larval habitats in Dire Dawa City Administration\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/9802313a33363d8c2020fdd0.png"},{"id":100938366,"identity":"201fe548-77ec-4ce0-9fc8-7bb55f1e697d","added_by":"auto","created_at":"2026-01-23 03:45:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41419,"visible":true,"origin":"","legend":"\u003cp\u003eMean \u003cem\u003eAnopheles\u003c/em\u003elarval density by month and ecological setting in in Diredawa, Ethiopia (2024)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/68b68491cc6bb24ea9a4491f.png"},{"id":100938367,"identity":"50d179e4-3daf-45c7-b85d-81f36d78a01d","added_by":"auto","created_at":"2026-01-23 03:45:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68130,"visible":true,"origin":"","legend":"\u003cp\u003eMean \u003cem\u003eAnopheles\u003c/em\u003elarval density by season, habitat type and ecological setting in Dire Dawa City Administration, Ethiopia (2024)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/4e381ebc77c5a9a3a88827ef.png"},{"id":100938374,"identity":"8b30163d-152e-4f92-a129-0fab2bad0be9","added_by":"auto","created_at":"2026-01-23 03:45:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101937,"visible":true,"origin":"","legend":"\u003cp\u003eVariables grouped by PCA, Variables grouped together means they are correlated. PCA 1 (red bars) includes \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eCulex \u003c/em\u003elarval presence, PCA 2 (green bars) includes \u003cem\u003eAedes \u003c/em\u003elarval density, PCA 3 (blue bars) includes \u003cem\u003eAn. stephensi\u003c/em\u003epresence\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/39d8c11b912cc2c34d45a121.png"},{"id":100952438,"identity":"1de3c65c-1c0f-412d-acaa-58de677179ab","added_by":"auto","created_at":"2026-01-23 07:16:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":301424,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Spearman’s correlation among predictors and occurrence of mosquito immatures.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/faf47eb09a812e910dabc57f.png"},{"id":100951814,"identity":"3ebce46a-e791-4bfb-aabd-4b4dc20f3b69","added_by":"auto","created_at":"2026-01-23 07:11:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":89603,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of Environmental and physicochemical parameters with presence and density of mosquito immatures.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/ef35dd103d328d01ab21c3c9.png"},{"id":100954572,"identity":"30e23bbb-6323-4478-9dc7-6bc140fe8af9","added_by":"auto","created_at":"2026-01-23 07:26:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3464298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8442371/v1/b77c5291-1655-495d-80f4-8ee2f02ef12b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anopheles larval ecology and physicochemical characterization of larval habitats in Dire Dawa: an area colonized by Anopheles stephensi in Eastern Ethiopia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAnopheles\u003c/em\u003e larval habitat characterization refers to the process of identifying and describing the environmental conditions where \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes lay their eggs and where their larvae develop [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These habitats are typically shallow bodies of water, such as ponds, swamps, streams, rice fields, and containers, which can vary in size and water chemistry. The characterization includes factors like water depth, vegetation, temperature, pH, salinity, and the presence of organic matter, as these conditions influence the survival and development of the larvae [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Understanding these habitats is crucial for malaria control, as targeting larval habitats with appropriate interventions, such as biological or chemical control methods, can help reduce mosquito populations and limit the transmission of malaria.\u003c/p\u003e \u003cp\u003eEach malaria vector has its own larval habitat preferences for oviposition that might be linked with their genetics make up or environmental parameters that imposed behavioral variation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main malaria vector \u003cem\u003eAn.gambiae\u003c/em\u003e complex prefers to breed in clean water of temporary rain pools, hoof prints, natural pockets of river, saline water (some species).The \u003cem\u003eAn.funestus\u003c/em\u003e groups breed in emergent vegetation and year round river sides, breeds in rice fields, and irrigation schemes. But the invasive \u003cem\u003eAn.stephensi\u003c/em\u003e breeds in artificial small containers, jars, bricks, plastic sheets, barrels, iron tankers, steel drums, used tire, ditches sometimes even in polluted water and fountains [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMosquito breeding habitats have specific biotic and abiotic factors that make them suitable to oviposition and support development of mosquito life cycle or metamorphosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies conducted in Ethiopia on \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. showed that the presence of predators, presence of other competing mosquito species, vegetation coverage and intervention, water temperature, electrical conductivity, pH, dissolved oxygen, salinity, total dissolved solution and turbidity are among the physicochemical factors that affect the density and distribution of mosquito larvae [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similar studies in India, Benin and Iran indicated the correlation between environmental factors and \u003cem\u003eAnopheles stephensi and other Anopheles spss\u003c/em\u003e larval abundance [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].But there are no studies that demonstrated the correlation between water physicochemical parameters and \u003cem\u003eAnopheles stephensi\u003c/em\u003e larval abundance in Ethiopia.\u003c/p\u003e \u003cp\u003eGlobally, LSM (larval source management) has a proven track record in malaria control and even elimination in some countries. Historical successes in Brazil, Egypt, and parts of Asia relied heavily on environmental management [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. More recent studies show that larviciding using biological agents like \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (Bti) can reduce larval and adult vector densities by up to 90% [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].In Ethiopia, larval source management is assumed to be a supplementary strategy besides the principal vector control interventions : use of insecticide treated nets and indoor residual spraying but there is inadequate larval identification and response to guide adoption of LSM approaches [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Ethiopia, pilot studies have shown promising results with significant reductions in larval density and adult vector population reduction through regular Bti and Sumilarv application [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] .Similarly, a study in Eastern part of the country on larval habitat suggested that LSM could be effective for focused control of \u003cem\u003eAn.stephensi\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, LSM integrated with LLINs and IRS resulted in a notable reduction in malaria incidence in Tolay area of oromia region and in northern Cot^e Idvore\u0026rsquo; ([\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrary, Ayana et al, 2025 reported that there was an increased malaria cases irrespective of LSM implementation and need to investigate factors that might affect the success of the intervention [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, this study was conducted to assess the physicochemical characteristics of larval habitats and relation with the ecology, season, larval density and distribution of larvae across the urban, peri-urban and rural areas of Dire Dawa.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was conducted in three ecological settings, of Dire Dawa City administration (515 Km East of Addis Ababa) which included urban, per-urban and rural settings. The urban setting considered for this study were Dire Dawa University, GTZ and Mermersa. The peri-urban sites were those around the industrial zone including villages of Melka-Jebdu and Jerba. The rural study sites were Aseliso, Hulahulul and Boren. The rural sites were agricultural sites with grass land and scanty vegetation coverage. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. During the study, larval source management was implemented jointly by the President\u0026rsquo;s Malaria Initiative (PMI) and the Ministry of Health of Ethiopia in the urban and peri-urban sites [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe study area has seasonal malaria transmission, with \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e the main parasite species,and the vectors include \u003cem\u003eAnopheles arabiensis\u003c/em\u003e and \u003cem\u003eAn. phareonsis.\u003c/em\u003e Recent studies reported the occurrence of \u003cem\u003eAn. stephensi\u003c/em\u003e as an additional vector [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmature mosquito collections and larval habitat characterization\u003c/h3\u003e\n\u003cp\u003eLarval surveys were conducted following standard WHO protocol and mosquito immatures were collected using larval dipper of 350ml capacity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Twenty dips were taken from each habitat by dipping at different sides of the habitat. Each larval instar and pupae were counted and recorded. Data were entered monthly using open data kit (ODK) immediately synced to the International Centers of Excellence for Malaria Research (ICEMR) database server to avoid data loss. Geographic coordinates, ecological setting (urban, peri-urban and rural), habitat type (Artificial and Natural) and picture of the habitat on spot, source of water (tap ,rain, or river), distance from household, land use (surrounding environment of the habitat), shade status/degree of exposure to sunlight, water flow, substrate type, distance from brick making, distance from nearest resting habitat and frequency of utilization, presence of canopy cover, presence of intervention, type of intervention and presence of predators were included among others. Mosquito immatures were classified as Anopheline, Aedine and Culicine based on morphological observations. The species composition of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were determined from adults reared from larvae or pupae.\u003c/p\u003e\n\u003ch3\u003eMolecular species identification\u003c/h3\u003e\n\u003cp\u003eThe collected adult \u003cem\u003eAnopheles\u003c/em\u003e were identified first to species using the morphological keys of Coetzee \u003cem\u003eet al\u003c/em\u003e. 2020 and Glich \u003cem\u003eet al\u003c/em\u003e. 1995 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Those identified as \u003cem\u003eAn. gamabiae\u003c/em\u003e s.l were further re-identified using species specific PCR. Real time PCR was also employed to confirm \u003cem\u003eAn. stephensi\u003c/em\u003e at the Genomics Laboratory of Jimma University Tropical Infectious and Diseases research center (TIDRC).\u003c/p\u003e\n\u003ch3\u003eDNA extraction and amplification\u003c/h3\u003e\n\u003cp\u003eDNA extraction was done using both the Chelex and Extracta protocols [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. PCR amplification was carried out according to the methods of Scott \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] using species-specific primers for \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003earabiensis\u003c/em\u003e (AR: 5\u0026prime;-AAG TGT CCT TCT CCA TCC TA-3\u0026prime;) and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eamharicus\u003c/em\u003e, formerly \u003cem\u003eAnopheles quadriannulatus\u003c/em\u003e B (QD: 5\u0026prime;-CAG ACC AAG AGA GAT GGT TAG TAT-3\u0026prime;)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eAnopheles gambiae\u003c/em\u003e (GA: 5\u0026prime;-CTG GTT TGG TCGGCA CGT TT-3\u0026prime;) and a universal primer (UN: 5\u0026prime;-GTGTGC CCC TTC CTC GATGT-3\u0026prime;). Then the amplicon was loaded on a 2% agarose gel stained with ethidium bromide and run for gel electrophoresis. \u003cem\u003eAnopheles arabiensis\u003c/em\u003e from the Sekoru insectary colony and \u003cem\u003eAn\u003c/em\u003e. \u003cem\u003eamharicus\u003c/em\u003e specimens from Arjo area were used as positive controls [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePhysico-chemical analysis of larval habitats\u003c/h3\u003e\n\u003cp\u003ePhysicochemical parameters of each habitat were measured using HANNA (H198184) instrument of multi-parameters (Multiparameter Meter, Hanna Instruments Inc., Woonsocket, Rhode Island, 02895, USA) after proper calibration of the instrument with the manufacturer\u0026rsquo;s probe solutions that was provided together with multi-parameter. Measurements of acidity and alkalinity (pH/mV), oxidative reduction potential (ORP), electrical conductivity (EC), total dissolved solids (TDS), resistivity, salinity, seawater, dissolved oxygen (DO), atmospheric pressure and temperature were taken from 102 larval habitats.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were entered in to SPSS version 27 and descriptive analysis was used to compare frequency, distribution, and magnitude of \u003cem\u003eAnopheles\u003c/em\u003e larvae in the urban, peri-urban and rural interfaces. Multiple regression analysis was used to compare the effect of physicochemical variables of habitats on the \u003cem\u003eAnopheles\u003c/em\u003e larva presence / absence, and larval density. Principal component analysis (PCA) was performed and Spearman\u0026rsquo;s correlation coefficient were used to dictate the correlation among water physicochemical parameters and larval presence and density. Cross tabs, Pearson\u0026rsquo;s Chi squared and Fisher\u0026rsquo;s exact tests were used to compare the association of presence/absence of predators, and presence/absence of intervention on larval presence and density. Larval density was calculated as number of larvae/pupae per 20 dips per habitat. Habitat index, container Index, and breteau Index were used to compare the larval density and abundance in each of the three ecological settings. Standard mathematical formulae [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] were used to calculate the larval indices as follows: 1) House Index (HI) was calculated by dividing the number of houses infested to the total number of houses inspected and multiplied by 100.\u003c/p\u003e \u003cp\u003e2) Container Index (CI) was calculated by dividing the number of containers positive for anopheles larvae to the total number of containers inspected and multiplied by 100. 3)Breteau Index (BI) was calculated by dividing the number of positive containers to total number of houses and multiplied by 100.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLarval habitat abundance and indices\u003c/h2\u003e \u003cp\u003eA total of 909 habitats were surveyed in Dire Dawa, of which, 857 (94.3%) were artificial breeding habitats such as bricks/concrete water tankers, plastic sheets, plastic water tankers (barrels), steel drums, canal ditches and tire tracks while the other were natural habitats such as river edges, animal hoof prints, ponds and swamps (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)( Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Dire Dawa, uncovered water tanker/Cemented cistern or brick were the predominant habitat type 520/909 (57.2%) and followed by plastic sheet 221/909 (24.3%). \u003cem\u003eAnopheles\u003c/em\u003e larvae were found in 297 (32.7%) of the houses surveyed and in 400(43.7%) of containers inspected in Dire Dawa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLarval habitat abundance, proportion and larval presence positivity in urban, peri-urban and rural sites in Dire Dawa City Administration (2024)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHabitat type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003en*(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive for Anopheles(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrick concrete water tanker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e520 (57.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e187(36.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDrainage ditch/canal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRiver edge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (2.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14 (56.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTire Tracks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (66.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFoot Print\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11(1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (81.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSwamp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (66.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePond\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSteel Drum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (2.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecovered drinking water tanker/Birca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 (6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (13.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlastic sheet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e221(24.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58 (26.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther (small water containers, pipe, foundation, septic tank)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (13.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e909 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e297 (33)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs depicted below in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, about 42, 34%, and 24% of larva habitats surveyed were from urban, peri-urban and rural settings respectively.\u003c/p\u003e \u003cp\u003eNine hundred-fifteen containers were found in houses surveyed with high number of containers 350(38.3%) in urban, 316 (34.5%) in peri-urban and 249 (27.2%) in rural sites respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLarval indices and the distribution of habitats in urban, peri-urban and rural ecologies in Dire Dawa City Administration, Ethiopia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHouses \u003c/p\u003e \u003cp\u003esurveyed\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e# positive\u003c/p\u003e \u003cp\u003e houses\u003c/p\u003e \u003cp\u003en (%)HI*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e#containers\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e#Positive \u003c/p\u003e \u003cp\u003econtainers CI*\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBreteau\u003c/p\u003e \u003cp\u003eIndex BI*\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e385 (42.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e133 (34.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e350 (38.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e175 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e175 (45.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeri-urban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e307 (34.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e107 (34.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e316 (34.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130 (41.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e130 (42.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e217 (24.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57 (26.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e249 (27.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95 (38.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95 (43.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e909 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e297 (32.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e915 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e400 (43.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400 (44)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eN\u0026thinsp;=\u0026thinsp;number, *HI-House index, CI:Container index and BI: Breteau index\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA total of 23,526 larvae and 1,808 pupae of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were collected from 909 habitats in urban, peri-uban, and rural areas of Dire Dawa. There was a difference in monthly larval density at each ecology and habitat type.\u003c/p\u003e \u003cp\u003ePaired-samples t-tests indicated that there is a significant difference in the density of \u003cem\u003eAnopheles\u003c/em\u003e larvae across months ( t=-3.27, n\u0026thinsp;=\u0026thinsp;34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;.003) with a mean difference of \u0026minus;\u0026thinsp;6.98 larvae per dip (95% CI: \u0026minus;11.32 to \u0026minus;\u0026thinsp;2.63) and a medium effect size (Cohen\u0026rsquo;s dₚ = 0.55), indicating a moderate seasonal influence on larval abundance. Similarly, the paired-samples t-test showed that larval density differed significantly between ecological categories ( t\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;5.37, n\u0026thinsp;=\u0026thinsp;34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.001) with a mean difference of \u0026minus;\u0026thinsp;11.52 larvae per dip (95% CI: \u0026minus;15.88 to \u0026minus;\u0026thinsp;7.16) and a large effect size (Cohen\u0026rsquo;s dₚ = 0.91). A stronger effect was observed across ecological settings, which suggests that ecological characteristics exert a greater influence on \u003cem\u003eAnopheles\u003c/em\u003e larval density than seasonal variation alone.\u003c/p\u003e \u003cp\u003eIn urban sites, where larval density was estimated to be constant throughout the year, the monthly \u003cem\u003eAnopheles\u003c/em\u003e larval density was high in October and lowest in May in urban settings. In peri-urban sites, \u003cem\u003eAnopheles\u003c/em\u003e larval density was highest in October and absent in May. In rural sites, larval density was peak in February and absent from June to October (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), showing that there was a significance difference between months and ecology, where some ecological settings were absent in some months (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.38, n\u0026thinsp;=\u0026thinsp;34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that temporal and ecological factors together influence larval dynamics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean larval density were highest in peri-urban site in artificial uncovered water tanker (51 larvae per dip), followed by natural ditch in urban site (46 larvae per dips), and lowest in rural and urban plastic sheet habitats, and lowest in plastic sheets in Urban sites (0 per dip/habitat).\u003c/p\u003e \u003cp\u003eHabitat type differs significantly across the season (t\u0026thinsp;=\u0026thinsp;6.36, n\u0026thinsp;=\u0026thinsp;77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) indicating seasonal variation affect habitat type. Anopheles larvae abundance and density were significantly different in different types of habitats (t = -4.8, n\u0026thinsp;=\u0026thinsp;77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There is a significant association between ecological settings (urban, peri-urban, and rural) and habitat types (t=-6.027, n\u0026thinsp;=\u0026thinsp;77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Seasonal variations, specifically dry and long rain seasons, strongly affect larval abundance and density (t = -4.95, n\u0026thinsp;=\u0026thinsp;77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eAs construction of houses and production of bricks are common in urban and peri-urban areas of Dire Dawa, \u003cem\u003eAnopheles\u003c/em\u003e larvae population density was high throughout the year in the artificial water storages, regardless of the seasonal variation in the urban and peri-urban ecologies. But in rural areas, \u003cem\u003eAnopheles\u003c/em\u003e larval density increased following the short rain season (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProximity of larval habitats to houses 80% (732 /909) were located in less than 50 meters from nearby living houses, 65% (589/909) of the habitats were at 0\u0026ndash;50 meters away from brick making houses. Additionally, 56% (509/909) of the habitats surveyed were surrounded by construction site or land-use for brick making. Half of the total habitats substrate were made of concrete with 35% (320/909) covered with plastic sheet or lining and about 11% of the habitat substrate was mud. The results of logistic regression analysis indicated that man-made/artificial habitat and grass land of surrounding were significant predictors for the presence of \u003cem\u003eAnopheles\u003c/em\u003e larvae/pupae (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLogistic regression analysis for the assessment of predictors of mosquito larval occurrence in urban, peri-urban and rural areas of Dire Dawa City Administration, Ethiopia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChiSquare\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProb\u0026thinsp;\u0026gt;\u0026thinsp;ChiSq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLower 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUpper 95%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEco setting[peri-urban]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.675\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEco setting[rural]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.747\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHabitat category[man made]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.805\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLanduse2[construction]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLanduse2[farmland]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLanduse2[grassland]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLanduse2[house]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.717\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.557\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLanduse2[road]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-1.943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-5.645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.139\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVegetation Cover\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShade coverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLSM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.540\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance to house\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance to resting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance to brick making\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHabitat length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3531\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBoth natural and man-made habitats were having same correlation with \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e larval presence and \u003cem\u003eAedes\u003c/em\u003e larval density. But rural sites were highly correlated with \u003cem\u003eAedes\u003c/em\u003e larval density than urban and peri-urban and sites. Conversely, \u003cem\u003eAn.stephensi\u003c/em\u003e larval presence were correlated with urban and peri-urban than rural sites. Similarly, the result of the PCA indicated positive correlation between land use (surrounding environment) and larval presence, habitat with in Grass-land environment, farm land, and availability of shrubs and in or near household were correlated with presence of \u003cem\u003eAnopheles, Culicines and Aedeines\u003c/em\u003e larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of principal component analysis (PCA) indicated that Larva/pupa presence were positively correlated with pH (r\u0026thinsp;=\u0026thinsp;0.264, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and water pressure (r\u0026thinsp;=\u0026thinsp;0.21, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, there was a significant positive correlation with temperature and conductivity (EC) (r\u0026thinsp;=\u0026thinsp;0.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), Total dissolved solids (TDS) ((, r\u0026thinsp;=\u0026thinsp;0.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) and, Salinity (r\u0026thinsp;=\u0026thinsp;0.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02)(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEC, TDS and salinity were positively correlated (r\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00). Also Dissolved oxygen (DO) and water pressure (psi) showed positive correlation (r\u0026thinsp;=\u0026thinsp;0.211, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040). But, temperature and resistivity had negative correlation (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.494, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00), pH and mvpH also had negative correlation (r= -0.993, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00). In addition, there was no significant correlation between temperature and Dissolved oxygen, Dissolved oxygen and Salinity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Oxydative reduction potential (ORP) was the variable that had no-significant correlation with the dependent variable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PCA result also showed that \u003cem\u003eAnopheles\u003c/em\u003e larval/pupa density were positively correlated with shorter distance to living house and distance to resting places, availability of brick making, vegetation cover, shading, pH and salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Pearson\u0026rsquo;s Chi squared test revealed a significance association between the predators and competitors presence on larval presence (ꭓ\u003csup\u003e2\u003c/sup\u003e=16.3, df:1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Larvae were found more often in locations where predators were present (66.7%) compared to locations without predators (31.5%).The result indicated that larva presence and predator presence are not independent, and predator and competitors presence is significantly associated with higher likelihood of larva presence.\u003c/p\u003e \u003cp\u003eBut, the Pearson\u0026rsquo;s chi squared test indicated that there is no significance association between presence of intervention (larviciding) and larval presence (ꭓ\u003csup\u003e2\u003c/sup\u003e =2.66, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.264), the presence of intervention doesn\u0026rsquo;t affect larval presence (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Clear water had positive correlation with presence of \u003cem\u003eAnopheles stephensi.\u003c/em\u003e Moreover, water salinity, water temperature and water pH were among the strong predictors of the presence of \u003cem\u003eAn.stephensi\u003c/em\u003e larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe analysis of water salinity revealed that the majority of samples (88.3%) fell within the valid or normal salinity range, indicating predominantly fresh water conditions across the study area. Only 11.7% of the total 873 samples exhibited measurable salinity above 0 ppt. Among these, slightly saline water (2\u0026ndash;3 ppt) was the most frequent, accounting for 5.6% of the samples, followed by low salinity levels between 0\u0026ndash;1 ppt (2.4%). Higher salinity categories were much less common, with only 1.8% of samples in the 4\u0026ndash;5 ppt range and less than 1% exceeding 6 ppt. Extremely saline samples (\u0026gt;\u0026thinsp;30 ppt) represented just 0.3% of the total (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of water salinity measurements of larval habitats in Dire Dawa City Administration, Ethiopia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalinity (ppt)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u0026ndash;1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21(2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVery low salinity (freshwater).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u0026ndash;3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49 (5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlightly saline (brackish tendency).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u0026ndash;5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModerately saline.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u0026ndash;7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNoticeably saline.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u0026ndash;9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHighly saline.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u0026ndash;14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVery saline\u0026mdash;approaching seawater levels.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (0.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtremely saline (seawater or hyper-saline).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe water physicochemical analysis indicated that water temperature of larval habitat ranged from 19\u0026deg;C to 34\u0026deg;C. The water temperature for 80.4% (82/102) of larval habitats was from 20\u0026ndash;27\u0026deg;C (room temperature), 16.6% (17/102) of the larval habitats water temperature was from 28\u0026ndash;34\u0026deg;C, and for the remaining 2.9% (3/102) of habitats, the water temperature recorded were 19\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe water pH values measured and categorized in to three as acidic water (pH value\u0026thinsp;\u0026lt;\u0026thinsp;7), neutral (pH value\u0026thinsp;=\u0026thinsp;7) and alkaline (pH\u0026thinsp;\u0026gt;\u0026thinsp;7).The result indicated that 86.3% (88/102) of habitats were alkaline, 12.7% (13/102) were neutral and 0.98% (1/102) of habitats were acidic.\u003c/p\u003e \u003cp\u003eFive types of substrate were considered (Gravel, Sand, Mud, Concrete and Plastic cloth) and the result of the study showed that 50% (52/102) of the habitats substrate were made of concrete and 25% (26/102) of the habitat substrate were made of plastic cloths/container. Fisher\u0026rsquo;s exact test indicated that there is a statistical association between larval presence and substrate type (ꭓ\u003csup\u003e2\u003c/sup\u003e=26.6, df\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSpecies diversity and evenness\u003c/h2\u003e \u003cp\u003eA total of 2,934 adult \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were emerged from immatures from all sites and \u003cem\u003eAn. stephensi\u003c/em\u003e was the predominant species 75% (2194/2934) followed by \u003cem\u003eAn.arabiensis\u003c/em\u003e 22% (636 / 2934), \u003cem\u003eAn. pharoensis\u003c/em\u003e 1.7% (50/2934), \u003cem\u003eAn. coustani\u003c/em\u003e 1.3% (38/2934), \u003cem\u003eAn.amharicus\u003c/em\u003e 0.3% (9/2934) and \u003cem\u003eAn. pretoriensis\u003c/em\u003e 0.2% (7/2934), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eAnopheles\u003c/em\u003e mosquito species diversity, richness and evenness by habitat type in Dire Dawa City Administration, Ethiopia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHabitat\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbundance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProportion\u003c/p\u003e \u003cp\u003e(pi)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLnpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epi*Lnpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eShannon \u003c/p\u003e \u003cp\u003ediversity index (H')\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSpecies \u003c/p\u003e \u003cp\u003eeveness (J): H/LnS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003cp\u003e richness (S)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eArtificial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.stephensi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.arabiensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.amharicus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.pretoriensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn coustani\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.pharoensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eNatural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.stephensi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.arabiensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.amharicus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.pretoriensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.coustani\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAn.pharoensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurther paired samples \u003cem\u003et\u003c/em\u003e-test revealed significant differences in species composition between habitat categories (man-made and natural). Abundance differed significantly between habitats (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2.22, n\u0026thinsp;=\u0026thinsp;13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), with man-made habitats having higher \u003cem\u003eAnopheles\u003c/em\u003e abundance. Significant habitat-related differences were also observed in Shannon diversity (t\u0026thinsp;=\u0026thinsp;10.03, n\u0026thinsp;=\u0026thinsp;13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), species evenness (t\u0026thinsp;=\u0026thinsp;5.70, n\u0026thinsp;=\u0026thinsp;13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and species richness (t\u0026thinsp;\u003cem\u003e=\u003c/em\u003e\u0026thinsp;5.7, n\u0026thinsp;=\u0026thinsp;13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating greater diversity, more even species distribution, and higher species richness in natural habitat category.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAn. stephensi\u003c/em\u003e, \u003cem\u003eAn.arabiensis\u003c/em\u003e and \u003cem\u003eAn.amharicus\u003c/em\u003e found to share same artificial habitats more specifically plastic sheets (in urban, peri-urban and rural), and in natural habitats more specifically a river Butuji river found in Dire Dawa city.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular identification of\u003c/b\u003e \u003cb\u003eAn.gambiae\u003c/b\u003e \u003cb\u003es.l and\u003c/b\u003e \u003cb\u003eAn.stephensi\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOf total 645 \u003cem\u003eAn.gambiae\u003c/em\u003e s.l sub-samples that were tested using PCR, 98.6% (n\u0026thinsp;=\u0026thinsp;636) were found to be \u003cem\u003eAn.arabiensis\u003c/em\u003e, and 1.4% (n\u0026thinsp;=\u0026thinsp;9) were \u003cem\u003eAn.amharicus\u003c/em\u003e. Similarly, real time PCR (qPCR) was performed to confirm morphologically identified \u003cem\u003eAn.stephensi\u003c/em\u003e specimens from \u003cem\u003eAn.gambiae\u003c/em\u003e s.l, and accordingly 200 sub-samples of each \u003cem\u003eAn.stephensi\u003c/em\u003e and \u003cem\u003eAn.arabiensis\u003c/em\u003e were proved distinct.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study indicated that \u003cem\u003eAn.stephensi\u003c/em\u003e immature stages are abundant throughout the year regardless of the seasonal variation of rain, with peak larval densities in dry season in all the three ecological settings. Similar results were reported by Yared \u003cem\u003eet al\u003c/em\u003e. 2023 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] in Somali region, Jigjiga town, where larvae were present in dry season. This might be because both Dire Dawa city and Jigjiga town have similar in expansion of urbanization and construction which support the breeding of immature stages in dry season.\u003c/p\u003e \u003cp\u003eMoreover, larval habitats were found in less than 50m from living houses and brick making houses were located at distance ranging from 0-50m away from living houses. This finding has an important implication as far as supporting the life cycle of \u003cem\u003eAnopheles\u003c/em\u003e and maximizing the probability of man vector contact. This finding could help to implement targeted larviciding, larval source management in habitats that harbor the immature stages throughout the year.\u003c/p\u003e \u003cp\u003eThe other finding of our study is the co-existence of \u003cem\u003eAn.stephensi\u003c/em\u003e with \u003cem\u003eAn.gambiae s.l\u003c/em\u003e, sharing same both artificial and natural habitats and ecological settings. \u003cem\u003eAn.stephensi\u003c/em\u003e were known to be vector of malaria mainly in urban settings and in an artificial habitats [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but in our study \u003cem\u003eAn.stephensi\u003c/em\u003e were found in rural ecology in Dire Dawa, and in river edges of Legehare and Butuji rivers, the two known and permanent habitats in the city. This further remarks the expansion of \u003cem\u003eAn.stephensi\u003c/em\u003e to rural sites and adapting natural habitats to breed. Our finding coincides with a study done in South east Iran where \u003cem\u003eAn.stephensi\u003c/em\u003e were found in river edges with sandy substrates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnexpected result of our research in the study area is, the presence of \u003cem\u003eAn.amharicus\u003c/em\u003e which were reported in South Western, Central, Western, [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and most recently in North west part of the country, here in the Eastern part of Ethiopia ,we report the existence of \u003cem\u003eAn.amharicus\u003c/em\u003e in urban,peri-urban and rural ecologies of Dire Dawa for first time. Additionally, \u003cem\u003eAn.coustani\u003c/em\u003e, \u003cem\u003eAn.pretoriensis and An.phareonsis\u003c/em\u003e were found in the study area.\u003c/p\u003e \u003cp\u003eThis finding recommends further investigation on the role of \u003cem\u003eAn.amharicus\u003c/em\u003e and the other vectors in malaria transmission in Dire Dawa and their general bionomics including its susceptibility to current insecticides in use are important.\u003c/p\u003e \u003cp\u003eWater physicochemical analysis indicted that larval distribution and abundance was not significantly different as the measures of the water physicochemical vary. Also a change in Ecology, season, habitat category doesn\u0026rsquo;t indicate a difference on presence and absence of larvae and density of larvae in man-made habitats. Our study align with the study conducted in Northern Iran which indicated no significance difference in physicochemical characteristics and larval density [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to our study, that indicated the analysis of water salinity revealed that the majority of samples (88.3%) fell within the valid or normal salinity range and the 86% fell in alkaline pH, indicating predominantly fresh water conditions across the study area, studies on correlation of \u003cem\u003eAnopheles\u003c/em\u003e larval density with physicochemical characteristics in Benin and South east Iran indicated that there was high positive correlation of larval density with temperature [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], dissolved oxygen and salinity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].This difference might be because of the difference in the source of water in urban areas of Benin and South east Iran from our study area where almost 98% of source of water for larval habitats were ground water and chemical characters were in similar ranges.\u003c/p\u003e \u003cp\u003eIn agreement with a study conducted by Mereta et al. 2013 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], our study found that there was wide spread distribution of \u003cem\u003eAnopheles\u003c/em\u003e larvae in small man-made aquatic habitats but in contrast to Mereta eta al. 2013 finding, in our study, \u003cem\u003eAnopheles\u003c/em\u003e larva was abundant in both natural and artificial habitats.\u003c/p\u003e \u003cp\u003eYear- round presence of larvae in artificial habitats indicated larval density, distribution and abundance were not affected by season as far as water supply continued for the purpose of mainly for construction and brick making. Expansion of \u003cem\u003eAn.stephensi\u003c/em\u003e to rural sites could be due to the rapid urbanization and industrial expansion which is accompanied with construction of water storages. The \u003cem\u003eAn.stephensi\u003c/em\u003e samples found in rural sites (40km away from Dire Dawa city) in Aseliso was in plastic sheet habitat type implicated that where ever the artificial habitats are used they are suitable for the exotic species to breed in. This study indicated the strong association of some ecological settings with the density and abundance of \u003cem\u003eAn.stephensi\u003c/em\u003e in urban and peri-urban than rural sites this could be due to availability of enormous construction and brick manufacturing in urban and peri-urban settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eAn.stephensi\u003c/em\u003e was the predominant species found in the study area, followed by \u003cem\u003eAn.arabiensis, An.amharicu, An.pharoensis, An.coustani and An.pretoriensis\u003c/em\u003e. Uncovered water tankers were the most efficient habitat types in urban and peri-urban sites followed by plastic sheet habitat types while hoof prints and river basins under natural habitats were the most efficient habitat types in rural and urban sites respectively. \u003cem\u003eAn. stephensi\u003c/em\u003e was found in rivers of urban site and plastic sheets of rural sites co-existing with \u003cem\u003eAn.arabiensis\u003c/em\u003e in same sites. Both shared same ecological settings and habitat types. \u003cem\u003eAn.amharicus\u003c/em\u003e was found to breed in artificial habitats of the three ecological settings.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe report here the existence of \u003cem\u003eAn.amahricus\u003c/em\u003e for the first time in the study area, Eastern part of Ethiopia, expansion of \u003cem\u003eAn.stephensi\u003c/em\u003e in rural areas, breeding in natural habitats, and co-existing with \u003cem\u003eAn gambiae s.l\u003c/em\u003e .\u003c/p\u003e \u003cp\u003eAvailability of brick making, shorter distance from living houses being in urban ecology were directly correlated with larval density. The result of our study revealed that there is significant correlation of ecological and seasonal variation in the density of \u003cem\u003eAnopheles\u003c/em\u003e larvae. Habitat abundance and positivity of uncovered water tankers in urban and pre-urban sites could indicate for feasibility and proper implementation of larval source management.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLimitation of the study\u003c/strong\u003e \u003cp\u003eThe physicochemical tests were performed in a cross-sectional way but we believe to be done during main rain season to observe if seasonal variation affects the water chemistry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study obtained financial support from the National Institutes of Health (D43 TW001505, R01 A1050243 and U19 AI129326). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEA, MB, GY and DY conceived and designed the study; EA, HM, HA and TD were \u0026nbsp;involved in data collection and field supervision. EA, TD and AA performed laboratory analysis. EA, ML, DZ and GZ performed the data analysis and EA drafted the manuscript. ML developed map of the study area and figures. TD, MB, DY and GY critically reviewed the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical clearance and amendment was obtained from the institutional review board (IRB) of the Institute of Health, Jimma University (Reference number) JUIH/IRB/236/25. Furthermore, permission was obtained from the Dire Dawa Health Bureau and health facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. Manual on practical entomology in Malaria. Part II. Geneva: World Health Organization; 1975.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson ML, Krogstad DJ, Arinaitwe E, Arevalo-Herrera M, Chery L, Ferreira MU, Ndiaye D, Mathanga DP, Eapen A. Urban malaria: understanding its epidemiology, ecology, and transmission across seven diverse ICEMR network sites. Am J Trop Med Hyg. 2015;93:110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilver JB. Mosquito ecology: field sampling methods. springer science \u0026amp; business media; 2007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGitheko A, Mbogo C, Atieli F. Resting behaviour, ecology and genetics of malaria vectors in large scale agricultural areas of Western Kenya. Parassitologia. 1996;38:481\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaenike J. Genetics of oviposition-site preference in Drosophila tripunctata. Heredity. 1987;59:363\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor R, Messenger LA, Abeku TA, Clarke SE, Yadav RS, Lines J. Invasive Anopheles stephensi in Africa: insights from Asia. Trends Parasitol. 2024;40:731\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalkew M, Mumba P, Yohannes G, Abiy E, Getachew D, Yared S, Worku A, Gebresilassie A, Tadesse FG, Gadisa E. An update on the distribution, bionomics, and insecticide susceptibility of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in Ethiopia, 2018\u0026ndash;2020. Malar J. 2021;20:263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eService M. Medical entomology for students. 2000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMereta ST, Yewhalaw D, Boets P, Ahmed A, Duchateau L, Speybroeck N, Vanwambeke SO, Legesse W, De Meester L, Goethals PL. Physico-chemical and biological characterization of anopheline mosquito larval habitats (Diptera: Culicidae): implications for malaria control. Parasit Vectors. 2013;6:320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsegaye A, Demissew A, Hawaria D, Abossie A, Getachew H, Habtamu K, Degefa T, Wang X, Lee M-C, Zhou G. Anopheles larval habitats seasonality and environmental factors affecting larval abundance and distribution in Arjo-Didessa sugar cane plantation, Ethiopia. Malar J. 2023;22:350.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas S, Ravishankaran S, Justin JA, Asokan A, Mathai MT, Valecha N, Thomas MB, Eapen A. Overhead tank is the potential breeding habitat of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in an urban transmission setting of Chennai, India. Malar J. 2016;15:274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoleimani-Ahmadi M, Vatandoost H, Zare M. Characterization of larval habitats for anopheline mosquitoes in a malarious area under elimination program in the southeast of Iran. Asian Pac J Trop Biomed. 2014;4:S73\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHessou-Djossou D, Dj\u0026egrave;gb\u0026egrave; I, Ahadji-Dabla KM, Nonfodji OM, Tchigossou G, Djouaka R, Cornelie S, Djogbenou L, Akogbeto M, Chandre F. Diversity of larval habitats of Anopheles mosquitoes in urban areas of Benin and influence of their physicochemical and bacteriological characteristics on larval density. Parasit vectors. 2022;15:207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeiser J, Singer BH, Utzinger J. Reducing the burden of malaria in different eco-epidemiological settings with environmental management: a systematic review. Lancet Infect Dis. 2005;5:695\u0026ndash;708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFillinger U, Kannady K, William G, Vanek MJ, Dongus S, Nyika D, Geissb\u0026uuml;hler Y, Chaki PP, Govella NJ, Mathenge EM. A tool box for operational mosquito larval control: preliminary results and early lessons from the Urban Malaria Control Programme in Dar es Salaam, Tanzania. Malar J. 2008;7:20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEthiopia FMoH. Ethiopia malaria elimination strategic plan: 2021\u0026ndash;2025. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeshome A, Erko B, Golassa L, Yohannes G, Irish SR, Zohdy S, Dugassa S. Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in Ethiopia. Malar J. 2023;22:48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYewhalaw D, Erena E, Degefa T, Kifle YG, Zemene E, Simma EA. Biological and residual activity of candidate larvicide formulation, SumiLarv 2MR, against an exotic invasive mosquito Anopheles stephensi Liston, 1901 (Diptera: Culicidae) in Ethiopia. Scie Rep. 2025;15:291.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYared S, Gebresilassie A, Aklilu E, Abdulahi E, Kirstein OD, Gonzalez-Olvera G, Che-Mendoza A, Bibiano-Marin W, Waymire E, Lines J. Building the vector in: construction practices and the invasion and persistence of Anopheles stephensi in Jigjiga, Ethiopia. Lancet Planet Health. 2023;7:e999\u0026ndash;1005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMutero CM, Okoyo C, Girma M, Mwangangi J, Kibe L, Ng\u0026rsquo;ang\u0026rsquo;a P, Kussa D, Diiro G, Affognon H, Mbogo CM. Evaluating the impact of larviciding with Bti and community education and mobilization as supplementary integrated vector management interventions for malaria control in Kenya and Ethiopia. Malar J. 2020;19:390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTia J-PB, Tchicaya ES, Zahouli JZ, Ouattara AF, Vavassori L, Assamoi J-B, Small G, Koudou BG. Combined use of long-lasting insecticidal nets and \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e larviciding, a promising integrated approach against malaria transmission in northern C\u0026ocirc;te d'Ivoire. Malar J. 2024;23:168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyana GM, Jalilian A, Ashine T, Molla E, Hailemeskel E, Yemane DH, Yirgu H, Negash N, Teferi N, Teshome D. Larval source management in Ethiopia: modelling to assess its effectiveness in curbing malaria surge in dire Dawa and Batu Towns. Malar J. 2024;23:366.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsri. ArcGIS Pro Version 3.5 [Computer software].Redlands,CA:Esri. (2025). 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e2023. TPVP: Ethiopia Final Entomological Report, April 2022-March 2023. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmiru T, Getachew D, Murphy M, Sedda L, Ejigu LA, Bulto MG, Byrne I, Demisse M, Abdo M, Chali W. Evidence for a role of \u003cem\u003eAnopheles stephensi\u003c/em\u003e in the spread of drug-and diagnosis-resistant malaria in Africa. Nat Med. 2023;29:3203\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerga H, Degefa T, Birhanu Z, Abiy E, Lee M-C, Yan G, Yewhalaw D. Urban malaria and its determinants in Eastern Ethiopia: the role of Anopheles stephensi and urbanization. Malar J. 2024;23:303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. Manual on practical entomology in Malaria. Part II. Methods and techniques. 1975.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrganization WH. Malaria entomology and vector control. In \u003cem\u003eMalaria entomology and vector control.\u003c/em\u003e 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoetzee M. Key to the females of Afrotropical \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes (Diptera: Culicidae). Malar J. 2020;19:1\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlick JI. Illustrated key to the female \u003cem\u003eAnopheles\u003c/em\u003e of southwestern Asia and Egypt (Diptera: Culicidae). Mosq Syst. 1992;24:125\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMusapa M, Kumwenda T, Mkulama M, Chishimba S, Norris DE, Thuma PE, Mharakurwa S. A simple Chelex protocol for DNA extraction from Anopheles spp. J Vis Exp 2013:3281.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott JA, Brogdon WG, Collins FH. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993;49:520\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoetzee M, Hunt RH, Wilkerson R, Della Torre A, Coulibaly MB, Besansky NJ. \u003cem\u003eAnopheles coluzzii\u003c/em\u003e and \u003cem\u003eAnopheles amharicus\u003c/em\u003e, new members of the \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex. Zootaxa. 2013;3619:246\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemissew A, Hawaria D, Kibret S, Animut A, Tsegaye A, Lee M-C, Yan G, Yewhalaw D. Impact of sugarcane irrigation on malaria vector Anopheles mosquito fauna, abundance and seasonality in Arjo-Didessa, Ethiopia. Malar J. 2020;19:344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilver JB. Mosquito ecology: field sampling methods. Springer; 2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemissew A, Animut A, Kibret S, Tsegaye A, Hawaria D, Degefa T, Getachew H, Lee M-C, Yan G, Yewhalaw D. Evidence of pyrethroid resistance in \u003cem\u003eAnopheles amharicus\u003c/em\u003e and \u003cem\u003eAnopheles arabiensis\u003c/em\u003e from Arjo-Didessa irrigation scheme, Ethiopia. PLoS ONE. 2022;17:e0261713.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikookar SH, Fazeli-Dinan M, Azari-Hamidian S, Mousavinasab SN, Aarabi M, Ziapour SP, Esfandyari Y, Enayati A. Correlation between mosquito larval density and their habitat physicochemical characteristics in Mazandaran Province, northern Iran. PLoS Negl Trop Dis. 2017;11:e0005835.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Physico-chemical characterization, mosquito larval habitat, Anopheles stephensi, larval density, Ethiopia","lastPublishedDoi":"10.21203/rs.3.rs-8442371/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8442371/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Understanding mosquito larval ecology \u0026nbsp;is essential for planning and implementations of vector control strategies. The biotic and abiotic factors affect larval occurrence , density, survival and morphogenesis of mosquitoes. Artificial containers are very suitable larval habitats for some species of Anopheles and Aedes mosquitoes in urban and peri-urban settings. Therefore, this study we identified, mapped and characterized larval habitats, estimated larval density and indices larval habitats. In addition, we determined the species composition of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes and species evenness in urban, peri-urban and rural areas of Dire Dawa city adminstration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Larval habitats were surveyed and identified monthly for a period of 16 months from February 2023 to December 2024 \u0026nbsp;in urban, peri-urban and rural areas of Dire Dawa. Mosquito larvae and pupae were collected and those larvae identified as \u003cem\u003eAnopheles\u003c/em\u003e were fed on fish-food. Emerged adults were provided with 10% sucrose solution and kept under standard conditions in field insectary. Females \u003cem\u003eAnopheles\u003c/em\u003ewas identified morphologically and further species-specific PCR assay was employed to identify members of \u003cem\u003eAn\u003c/em\u003e.\u003cem\u003egambiae\u003c/em\u003e s.l. In addition, real time PCRassay was performed to identify \u003cem\u003eAn.stephensi\u003c/em\u003eand \u003cem\u003eAn.arabiensis\u003c/em\u003e. Water samples were taken from the larval habitats and the physico-chemical parameters were measured using HANNA Multi-parameter (H198194). Larval habitat diversity, larval abundance and distribution were assessed across the three ecological settings (urban, peri-urban and rural).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: A total of 23, 526 larvae and 1,808 pupae of \u003cem\u003eAnopheles \u003c/em\u003emosquitoes were collected from 909 man-made(uncovered cemented cisterns (Brick), plastic sheets, steel drums, Tire tracks, Canal ditch, plastic tanker/Barrel) and natural habitats (River edges, ponds, animal hoof prints and swaps) in urban, peri-urban areas of Dire Dawa. The highest mean larval density (51 larvae per dip) of \u003cem\u003eAnopheles\u003c/em\u003emosquitoes was recorded from peri-urban sites in uncovered water tanker (brick) followed by urban site in brick (46 larvae per dip). \u003cem\u003eAnopheles\u003c/em\u003e larvae were not found in steel drum and plastic barrels in rural sites.\u003c/p\u003e\n\u003cp\u003eA total of 2,934 adult \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were emerged from immatures collected from all sites, of which 75% (2194/2934) were \u003cem\u003eAn. stephensi\u003c/em\u003e, and 22 % (636 / 2934) were \u003cem\u003eAn.arabiensis\u003c/em\u003e. The remaining 3.0% were \u003cem\u003eAn. pharoensis\u003c/em\u003e, \u003cem\u003eAn. coustani\u003c/em\u003e,\u003cem\u003e An .amharicus,\u003c/em\u003e and \u003cem\u003eAn. pretoriensis.\u003c/em\u003e \u003cem\u003eAnopheles stephensi\u003c/em\u003e, \u003cem\u003eAn. arabiensis\u003c/em\u003e and \u003cem\u003eAn. amharicus\u003c/em\u003e shared the same habitats across the three ecologies. Larval density was positively correlated with availability of brick making, proximity to houses, \u0026nbsp;urban setting, presence of competitors /predators, vegetation cover, shade cover and substrate type. But larval presence was not correlated with presence/absence of intervention. Larva/pupa presence were positively correlated with pH (r=0.264, \u003cem\u003ep\u003c/em\u003e=0.01) and water pressure (r=0.21, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).There was positive correlation among temperature, electrical conductivity (EC), total dissolved solids (TDS), salinity and dissolved oxygen (DO) and negative correlation among temperature with resistivity, pH with mvPH. Larval presence was positively correlated with water salinity and pH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003e\u003cem\u003eAnopheles stephensi\u003c/em\u003e was the predominant species found in the study area, followed by \u003cem\u003eAn.arabiensis,\u003c/em\u003e \u003cem\u003eAn.amharicu, An.pharoensis, An.coustani and An.pretoriensis\u003c/em\u003e. Uncovered water tankers (Bricks) were the most prolific artificial habitats in urban and peri-urban sites followed by plastic sheets while natural habitats such as hoof prints and river basins were the most efficient habitat types in rural and urban settings, respectively. \u003cem\u003eAnopheles stephensi\u003c/em\u003e was found in natural habitats of Butuji and legehare rivers from urban site, and in rural sites from man-made habitat of plastic sheet.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnopheles amharicus\u003c/em\u003e larvae was found in plastic sheet, an artificial habitat common in urban, peri-urban and rural areas.\u003c/p\u003e\n\u003cp\u003eWe report here the occurrence \u0026nbsp;\u0026nbsp;of \u003cem\u003eAn.stephensi\u003c/em\u003ein rural areas, breeding in natural habitats, and co-existing with \u003cem\u003eAn. gambiae s.l\u003c/em\u003e complex.\u003c/p\u003e\n\u003cp\u003eAvailability of brick making, shorter distance from living houses being in urban ecology were directly correlated with larval density. Habitat abundance and positivity of uncovered water tankers (cemented cisterns or bricks) in urban and pre-urban sites could indicate for feasibility and proper implementation of larval source management.\u003c/p\u003e","manuscriptTitle":"Anopheles larval ecology and physicochemical characterization of larval habitats in Dire Dawa: an area colonized by Anopheles stephensi in Eastern Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 03:45:28","doi":"10.21203/rs.3.rs-8442371/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T00:55:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T21:11:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T08:22:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T14:31:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192281035899340420695375309360032029151","date":"2026-02-18T15:32:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159430930080569351203297855765050424608","date":"2026-02-18T06:48:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T16:17:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94195762604399300640684750369664368581","date":"2026-02-16T07:51:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25211909495416616501129529321539090128","date":"2026-02-16T05:45:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156506519611263394901471466319987410733","date":"2026-01-28T07:53:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T02:04:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T04:36:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-24T18:14:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-12-24T11:28:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17f8728c-dbf6-46f7-bae6-d563178025d1","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T00:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 03:45:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8442371","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8442371","identity":"rs-8442371","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}