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.

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.

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 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].

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.

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.

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.