Study Area
The study was carried out in Mutare City located at a latitude of 18o5'0" and longitude 32o40'0" (estimated population: 262 124) (Fig. 1). Mutare is Zimbabwe’s fourth-largest city located 265 kilometers to the east of Harare and lies north of the Vumba Mountain ranges that extend into Mozambique. The city covers an area of 191km2 and has a population density of 981.3/km2. It has a temperate climate with an average annual temperature of 19°C and moderate altitude (1110 m). The rains in the area are mostly received in the months of mid-November to April each year and the city receives more than 1000 mm of rainfall annually. Mutare City has a high groundwater table and is dotted with marshes, swamps, and ponds, combined with disturbances resulting from human activities, provides excellent breeding habitats for mosquitoes. The local ecological features of the area are predominantly savanna grassland and woodland ecosystem and the selection of breeding sites was mainly based on the ecological condition known to be potential breeding habitats for the vector. The study leveraged on the existence of laboratory and insectary facilities at Africa University and an already assembled field team of Scouters as well as community and logistical support from the Mutare City Council.
The area along the Zimbabwe-Mozambique border continues to bear the biggest burden of malaria [27]. This porous frontier lacks defined demarcations between the two countries and citizens from either side crosses the borders easily without formal documentation. This causes an uncontrolled entry and exit of traders between the two countries making it difficult to control the disease. Zimbabweans frequently visit Mozambique to buy goods for resale while Mozambicans cross into Zimbabwe in search of casual opportunities in commercial farms [28]. A study by Chiruvu et al., (2017) revealed that the proportion of patients from Mozambique that were treated for malaria in Zimbabwean health facilities increased from 2013–2015 [28]
Study Period
The collection and rearing of mosquito larvae were done during the dry season from 1 June to 5 November 2019 that is from winter up to the end of the dry season.
Entomological Sampling
The researchers stratified Mutare City into Northern, Southern, Eastern, and Western Regions. The four regions had different population densities with the Western and Southern regions experiencing dynamic changes in demography due to unplanned and uncontrolled urban expansion. Trained mosquito breeding site scouting experts employed by the Mutare City were employed to identify the potential vector breeding habitats. The Scouters also resided in their respective suburbs and had good knowledge of the area. Each of the four regions was visually inspected for the presence of water bodies and what influenced their creation. At least three Scouters did a thorough search of outdoor potential breeding sites for each residential suburb. These potential breeding habitats for the different regions were further stratified into man-made artificial (e.g. construction, earth mining, leaking pipe sites, storage containers, ponds) or natural origin (e.g. streams, dams, swamps) aquatic habitats. The breeding habitats for each stratified region were noted down as the sampling frame for that area and each breeding habitat was allocated a computer-generated number. All mosquito breeding habitats were identified with GPS coordinates of the site before being recorded on the survey form. A potential vector breeding habitat was defined as an accessible stagnant open water source conducive to Anopheline larval breeding.
Table 1
Mutare City stratification by region.
Stratification (Regions)
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Residential suburbs
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Northern Suburbs
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Murambi, Fairbridge Park, Tiger's Kloof, Avenues, Yeovil, Westlea, Florida, Central Business District
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Southern Suburbs
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Sakubva; Dangamvura, Weirmouth; Fern Valley, Darlington, Nyakamete Industrial Area, Muneni, Gimboki
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Eastern Suburbs
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Bordervale, Morningside, Palmerston, Greenside
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Western Suburbs
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Utopia, Chikanga, Hob-House, Natview Park; Zimta Park
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Sampling size determination of the potential breeding habitats took environmental heterogeneity into consideration. The target sample size was determined using Fisher’s formula. For equal allocation of site, a design effect of 3 [30] was utilized in calculating the minimum sample size. This study also used the most conservative estimate of p (0.5) [31], an alpha level of 95% (1.96), a maximum tolerable error of 10%, and a precision of 0.05 in sample size calculation. Thus, n = 3(1.96)² x 0.5(1- 0.5)/ 0.10² yielded an average sample size of 288 breeding sites. This study used an acceptable standard deviation of 23% and the ultimate sampling range was therefore 66 ± 288.
Habitat Characterization
The potential larval habitats were extensively searched for mosquito larval stages, specifically for immature aquatic stages. The habitats were characterized by coverage of canopy, surface debris, emergent plants, water depth, and habitat types. Sampled habitats without larvae present were recorded as “negative habitats”, those with larvae were recorded as “positive habitats”. Habitats with perimeters larger than 10 m were sampled at multiple stations between 5 m apart. For such habitats, only one geolocation of the sampling stations was recorded to represent the whole habitat. Environmental parameters included abiotic and biotic categories were further defined by sub-categories provided by a table. Habitats were classified into man-made and natural sites. The sampling and characterization of the potential larval habitats were done twice for bodies around residential suburbs if the first survey showed no results and the station was a well-known hotspot of invasive species from previous collections.
Abiotic characteristics included the water depth -shallow (< 1 meter deep), deep (≥ 1 meter deep); Sunlight- semi-shade, shade, deep shade; debris present or absence, and type of breeding site- river-bed, stream, dam, swamp-like seepage, hoof print, rain pool. Biotic parameters were classified by the canopy (vegetation above habitats), vegetation (living within the aquatic habitat), and these vegetation classifications were-none, floating vegetation, and emergent (emergent plants, floating, none). To maintain consistency, all visual classifications were done by one person who was also the principal investigator.
Collection and rearing of mosquitoes larva
A standard larvae collection form was used while geocoding and site description was done for each selected breeding site. The standard dipping method [32] was used to collect mosquito larvae utilizing 250 ml dippers times 10 dips taken at each habitat station. The mosquito larvae were placed in plastic jugs soon after larval density assessment and morphological determination of species. Anopheline larvae were separated from Culicines. The jugs were transported to the Africa University insectaries where they were reared according to the WHO (2003) guidelines [33]. The larvae were pooled according to region and habitat where they were obtained from. The mosquito larvae were screened morphologically and counted using taxonomy keys of Gillies and De Meillon [34]. Collected larvae were raised to adults and live Anopheles mosquitoes were killed by freezing before identification to species level by using morphologic keys developed by Gillies and Coetzee [35]. For post morphological identification, the mosquito samples were preserved individually in vials with silica gel for molecular differentiation of species.
Data analysis
Obtained data were entered and analyzed using Epi Info version 7.2.1.0 statistical package.