Study area
This study was carried out in Khartoum in central Sudan, at the confluence of the White Nile and Blue Nile, (31.5–34°E and 15–16°N), over 250 km and a total area of 20,736 sq km (Fig. 1). According to the Sudan Meteorological Authority (SMA), the temperatures range between 25 and 40 °C during the months April to June and between 20 and 35 °C during July to October. Temperatures fall during the winter period between November and March to 15–25 °C.
Most of Khartoum State falls within the semi-desert climatic zone while the northern part falls within the desert climatic zone. The State is prevailed with a hot to very hot rainy season during the summer and warm to cold dry season during the winter. Rainfall ranges between 100 and 200 mm in northeastern parts, 200–300 mm in southern parts and 10,100 mm in the northwestern parts.
Khartoum State has a population of 5,271,321, according to the Census conducted in 2008 by the Central Bureau of Statistics [10]. Khartoum’s drainage and sanitation systems are in poor condition. Only 28% of Khartoum is connected to a sewage system, and most residents use pit latrines and other basic systems such as septic tanks and siphon wells.
Larval surveillance
Two cross-sectional larval surveillance surveys were carried out in 5 localities in Khartoum State: Khartoum, East Nile, Jubal Aulia, Khartoum North, and Omdurman. The first survey was carried out during the hot dry season from February to April 2015 and was designed to study the possibility of Anopheles larvae to breed in polluted aquatic habitats. The second surveillance was conducted during the late rainy season from September to November 2015 to examine the susceptibility of An. arabiensis found in polluted water bodies to Temephos larvicide.
Survey 1
Larvae specimens were collected from only apparently polluted habitats after visual inspections. According to [3, 6], polluted larval habitats of An. gambiae s.l. are: “semi-permanent water collections containing domestic waste or organic products in decomposition that could be invaded by moisture and/or algae”. The expectation of polluted larval habitats in urban areas came after: visual inspection (colour and odour); presence near drainage system; presence near industrial areas presence near effluent from local houses and presence near university campus and military camps.
A handheld global positioning system (GPS) was used to determine and record the precise grid co-ordinates of the positive larval habitats. Samples of Anopheles larvae present in suspected polluted habitats were collected using standard dipping methods and preserved in 80% ethanol in small glass bottles with full labels. Water samples from each site were collected concurrently to study physiochemical parameters and preserved in clean plastic bottles (600 ml).
Survey 2
Mosquito larvae were collected from 4 localities in Khartoum State: Khartoum, Khartoum North, East Nile, and Jabal Awlia, to determine susceptibility of An. arabiensis larvae in polluted larval bodies to Temephos larvicide. Samples were collected from two types of habitats:
-
Polluted Anopheles species habitats that represented the positive Anopheles larvae habitats identified during Survey 1.
-
Non-polluted Anopheles species larval habitats in agricultural areas within urban Khartoum State.
Collection, preservation and identification methods
Anopheles larvae were sampled using standard dipping method; from each breeding site, 10 dips were taken with a standard 300-ml dipper according to [11]. Larvae were preserved in 80% ethanol in glass bottles. In the laboratory, larvae were identified morphologically using [12] and molecularly using species-specific identification according to [13]. Melanic and normal forms were classified according to [14].
Physicochemical analysis of water samples
Water samples were collected from each visually polluted larval habitat in 600-ml clean plastic bottles. To study the magnitude of water pollution at the breeding sites eight abiotic factors were determined, including colour, odour, pH, conductivity, total dissolved solids, turbidity, oil and nitrate. Water pollution associated with heavy metals was determined for three heavy metals (Cu, Pb, Fe), which were chosen as they were previously studied in An. gambiae s.s. polluted larval habitats [1]. Water samples were analysed in the Central Laboratory, Chemistry Department, Khartoum University. pH, conductivity and total dissolved solids (TDS) were analysed using pH 315i/SET, NO: 2A10-1012 and Cond 315i/SET, NO: 2C10-001, Germany, and turbidity was measured using Palintest: Turbimerer, PT 0900513197, UK. Heavy metals were determined using atomic absorption spectrophotometry at the Central Laboratory, Chemistry Department, Khartoum University. Physiochemical parameters from apparently polluted larval habitats were compared to physiochemical parameters of a control larval habitat (an irrigation canal at an agricultural area within Khartoum State).
Bioassay tests
The susceptibility bioassays were performed according to [15]. Twenty-five late third to early fourth-instar larvae of An. arabiensis were laboratory assayed for sensitivity to Temephos larvicide using four diagnostic concentrations; 0.005, 0.025, 0.125, and 0.625 mg/l. The average temperature of the water was 25 °C. Each concentration was replicated three times. After a period of 24 h, mortality counts were performed. Control trials were performed under same conditions.
Data analysis
Data were analysed using SPSS version 15.0. Descriptive statistics were used. The relationship between habitats variables and presence of anophelines in polluted water was tested using correlation. Only variables with P values < 0.05 were considered. The results of the insecticide susceptibility tests were analysed for dose/response relationship using regression probit analysis [16]. The 50% lethal concentration or LC50 value is commonly accepted as the basis for comparison in the investigation of relative toxicities among insecticides used.