Background

Mosquitoes are small two-winged biting insects belonging to family Culicidae, order Diptera. The family Culicidae comprises at least 3531 species representing 112 genera divided into two subfamilies, Culicinae and Anophelinae (Harbach, 2013). Three genera of medically important mosquitoes are found in Sudan, Anopheles, Culex and Aedes. One hundred and fifty six species and two subspecies of Culicidae have been recorded in Sudan (El Rayah, 2007).

Mosquitoes affect humans and animals by transmitting the disease-causing agents of several serious diseases, namely protozoans, arboviruses and filarial worms. Malaria is transmitted exclusively between human through bites from adult female mosquitoes of the genus Anopheles (Sathe and Tingare, 2010; Choumet, 2012). Mosquitoes of the genus Culex transmit viral diseases like Yellow fever, West Nile Viruses and Rift Valley fever and the genus Aedes transmits both Dengue fever as well as Rift Valley fever (Favier Charly et al., 2006). Filarial worm is transmitted by three mosquito genera: Anopheles, Culex and Aedes (Kemenesi et al., 2015).

In Kassala State, eastern Sudan malaria represents the major threat to public health (WHO, 2011). A mosquito-borne viral diseases, Dengue Fever and Rift Valley fever have been reported in recent years from the state (Ministry of Health, Kassala State, 2015). The presence of these mosquito-borne diseases in Kassala State indicates the importance of investigation the ecology of mosquitoes in this region of Sudan.  The major mosquito vectors in eastern Sudan are Anopheles arabiensis (Hamza et al., 2014), Aedes aegypti (Abdalmagid and Alhusein, 2008), Culexpipiens, and Culex quinquefasciatus (Lewis, 1945).

Mosquito-transmitted diseases can be controlled by interrupting or interfering with its transmission. Several measures are used to control these diseases including mass chemotherapy and control of the mosquito species. Of all methods used, control of mosquitoes is the best approach for protecting man and his domestic animals against mosquito-borne diseases. A fundamental understanding of the distribution and ecology of mosquito larvae is essential for effective vector control strategies (Mereta et al., 2013). Successful larval control requires a good knowledge of the breeding ecology of mosquitoes including, types and preferences for larval habitats, distribution of breeding sites, biological and physicochemical characteristics of the habitats (Olayemi et al., 2010).

Mosquito species differ in the type of aquatic habitats they prefer for oviposition based on location, physicochemical conditions of water body, and the presence of potential predators (Shililu et al., 2003; Piyaratnea et al., 2005). Physiochemical compositions of the water bodies are complicated and determine their condition and fauna composition. Physicochemical factors that influence oviposition, survival rate and the distribution of mosquito species include salts, dissolved organic and inorganic matter, degree of eutrophication, turbidity, presence of suspended mud, temperature, light, shade, Hydrogen ion concentration and presence of food substances (Okogun et al., 2005; Ephantus, 2008). Larval control can be achieved through larval habitat management by altering physiochemical properties of breeding habitats (Minakawa et al., 1999; Yasuoka et al., 2006; Chatterjee et al., 2015). Understanding the impact of these parameters will lead to better decision making in relation to mosquito control activities.

A previously published study by Hamza and Rayah (2016) described the types of Anopheles mosquito larval habitats in Kassala town, eastern Sudan in qualitative evidence without measuring larval habitat productivity. In the current study we investigated larval habitat productivity in relation to physicochemical water parameters in one type of mosquito larval habitats described by above two authors, We selected man-made ditches (about 300- 2000 m² surface area) created by human activities for building purposes and associated with rainwater as a source of water during the rainy season. These water bodies were scattered in the area and support mosquito breeding during and after the rainy season. Therefore, the current study designed to investigate this type of larval habitats to assess the mosquito immature stages abundance, evaluate the physicochemical characteristics that govern the presence of mosquito larvae, and determine the relationships between such characteristics and immature stages abundance in the area. The study provides information on mosquito ecology in relation to prevailing physicochemical characteristics of breeding habitats, which may have implication for vector distribution in the area.

1 Materials and Methods

1.1 Study area

This study took place in Kassala town (15° 28′ N latitude and 36° 24′ E longitude), eastern Sudan (Figure 1), covering an estimated area of 4030 K m² with a projected population of 298,529 million people with 2.8% annual growth rate from 2008 population census. It is located under arid-semiarid climate characterized by a short rainy season (July-October) followed by a cool dry season (November–February) and then a hot dry season (March-June). August with an average rain fall of 148 mm is the peak of the rainy season (Ministry of Energy and Mining, Sudan, 1989). The topography of the area is nearly flat characterized by the presence of backbones of mountains and seasonal small streams running during the rainy season. Soils mainly consist of clay, sandy clay, slit (Sudan, Town Planning Department and Netherlands, 1979).

For this study an initial visual investigation was carried out at the beginning of the rainy season in August 2016 to identify every human-made ditches filled with rain water that can serve as mosquito breeding sites and would be remain for long-term study. Subsequently, the town was divided in to three sections and each section was represented by two larval habitats. Thus, a representative sample of six aquatic larval habitats was selected as fixed places for mosquito collection. The selected larval habitats represent the different types of soils found in the area, two sites from western Kassala located at the basement of Kassala Mountain with sandy substrate. The other 4 sites, two from central and two from western Kassala were located in plain area with muddy substrate (Figure 2). Geographic coordinate readings (longitude and latitude) of all selected sampling sites were recorded using a hand-held global positioning system unit (GPS) (Garmin, etrex-VISTA HCX). These habitats were then plotted on a map of Google earth using ArcMap 9.3 software.

1.2 Specimens collection and identification

Larval specimen collection was carried out monthly in the larval habitats during the short rainy season from September to November 2016. The larval habitats were sampled for mosquito larvae using standard dipper (11 cm diameter and 200 ml capacity) according to WHO (1975). Samplings were always done by the same individuals in the morning (10: 00 -12:00 pm) for about 40 min at each larval habitat. Dipping took place where mosquito larvae were expected (edges of sites, hoof prints, around vegetation, shallow areas, etc). When mosquito larvae were present, 30 dips were taken, poured in suitable plastic container (about 10 liters capacity) and transported to the Zoology Laboratory at Kassala University, Kassala town. In the laboratory, water collected by dippers was emptied into a white sorting tray and all aquatic forms of mosquitoes (larvae and pupae) were sorted out, counted and identified into genera by morphological criteria as described by Hopkins (1952), Gillies and De-Mellion (1968) and Gillies and Coetzee (1987).

1.3 Physicochemical analysis of mosquito breeding habitats water

Simultaneously with larval sampling, physical parameters of the larval habitats including intensity of light, water current and turbidity were estimated by visual inspection and recorded. Intensity of light was categorized as full sunlight, partial sunlight and shade. Water current, was estimated as stagnant, slow flowing or high flowing, whereas turbidity was classified as clear, turbid and foul. Water temperature was determined at the field before larval collection using a thermometer.

During larval sampling, water sample was collected from each larval habitat in a plastic bottle (750 ml capacity), tightly closed and labeled with date of collection and habitat number and transported to the laboratory for analysis.

Water sample was analyzed for the following chemical characteristics: electrical conductivity (EC), total dissolved salts (TDS), pH, total hardness, total alkalinity and ions such as chloride (Cl^-), calcium (Ca⁺⁺), magnesium (Mg⁺⁺), sulphate (SO⁺⁺₄), fluoride (F^-) and nitrates (NO^-₃). Electrical conductivity was measured using conductivity meter (CD M210). Total dissolved salts were calculated based on conductivity value. PH was measured using pH meter (pH M210) while total alkalinity, chloride and total hardness were measured via titration. Sulphate, fluoride and nitrates were measured using spectrophotometer (Dril 5000). Values of chemical parameters were compared to standard values of drinking water quality according to the WHO (1993).

1.4 Statistical analysis

Data obtained from analyzed water samples were subjected into SPSS Version 16 and then were analyzed. The densities were calculated as number of mosquito larvae per dip. Descriptive analysis was done to calculate the mean values of water parameters. Monthly variation in larval abundance and chemical parameters in sampling larval habitats was determined using one-way analysis of variance (ANOVA) test. The difference in the abundance of the two mosquito genera, Anopheles and Culex was examined using T. test. Person correlation analysis was used to assess the relationship between larval abundance and individual chemical parameters of the larval habitats.

2 Results and Discussion

2.1 Occurrence of mosquito immatures

Table 1 shows the monthly number of mosquito immature stages collected from six representative larval habitats in Kassala town, eastern Sudan during the short rainy season 2016. During this larval sampling, a total of 5020 mosquito larvae and pupae were collected. Two mosquito genera were morphologically identified. Anopheles was the most abundant, representing 99.26% (n=4983) of the total, while Culex was found in low densities 0.74% (n=37). There is a significant difference in the abundance of the two mosquito genera encountered in the study (p < 0.05).

We did not found mosquito aquatic stages during the preliminary investigation of larval habitats at the beginning of the rainy season, in August. ANOVA test showed the monthly dynamics of mosquito aquatic stages in the studied larval habitats. Anopheles was found in high densities over the period of the study with a peak in November, following the short heavy rainy season (12.37 per dip), when approximately 37.23% were collected. The differences in densities were not significant among months (F =0.436; p > 0.05). Culex mosquitoes were found only in larval stages with significant variation among months (F=6.212; p< 0.05). Culex larvae were almost exclusively found in September- mid rainy season- where 97.30% of the total were collected in low densities (0.20 per dip) and least in October (0.01 per dip). No significant association was found between the abundance and density of Anopheles and Culex aquatic stages (r=-0.05; p > 0.05).

In this study we observed co-occurrence of Anopheles and Culex mosquitoes in the selected larval habitats and they use similar water collection to breed. Similar observations in an irrigated area, New Halfa town in eastern Sudan were reported by Himeidan and El Rayah (2008). Previous work reported the co-occurrence of these two genera in rural western Kenya (Minakawa et al., 1999; Fillinger et al., 2004) and in rice plots and irrigation wells in western Côte d’Ivoire (Matthys et al., 2006).

2.2 Physicochemical characteristics of larval habitats

Table 2 summarizes the monthly chemical characteristics of the selected larval habitats during the rainy season 2016. All of the measured chemical parameters were found in low concentrations except fluoride (1.12±0.45 mg/L) and calcium (24.47±3.23 mg/L) which were found in comparatively moderate concentrations.

The monthly mean values of measured chemical characteristics did not vary significantly except for the following parameters: pH (F=13.438; P<0.05), calcium (F=5.590; P<0.05) and magnesium (F=9.488; P<0.05). The mean pH values observed in this study were always within the alkalinity pH range (7.60-9.00) confirms previous observations made in the town that larval habitat of the mosquito Anopheles arabiensis showed a pH value of 7.9. Larvae of anopheline species mostly prefer habitats with neutral pH to slightly alkaline environments as observed by many workers in the world (Abdullah et al. 1995; Piyaratnea et al., 2005; Kudom et al., 2012; Liu et al., 2012; Soleimani-Ahmadi et al., 2014). On the other hand our findings disagree with Adebote et al. (2008) who reported the preference of Anopheline species to low pH values, acidic nature.

Correlation between larval abundance, i.e. the monthly number of mosquito aquatic stages sampled per larval habitat, and mean values of chemical characteristics of larval habitats revealed that occurrence of Anopheline aquatic stages was only associated, positively, with nitrates (r = 0.53; P<0.05) confirms previous observations reported by Mala and Irungu (2011). Our results disagree with Edillo et al. (2006) who reported that nitrates had no effect on abundance and forms of Anopheles mosquito species in a Malian village. Ndenga et al. (2012) reported the presence of Anopheline larvae in aquatic habitats with highest levels of nitrates of (10- 20 mg/L) with a mean of 12.0. In contrast, our study found nitrates in low concentration (5.57±3.71) in the larval habitats indicating little organic pollution attributed mainly to contamination from human and domestic animal faecal matter (Reisen, 2001).

Correlation analysis revealed that presence of Culex larval stages was associated, negatively with pH value (r = -0.56; P<0.05) and positively, with sulphate (r= 0.56; P<0.05). Our findings disagree with the positive association of pH value with culicines larval presence (Burke, 2010; Tadesse et al., 2011; Stein et al., 2011; Soleimani-Ahmadi et al., 2014). Sattler et al. (2005) reported that a pH value of less than 7.3 was associated with high culicine larvae. In contrast, our study found low Culex larval densities in larval habitats with pH values of 7.60-9.00.

These correlated characteristics were found to be the key factors which are associated with occurrence and abundance of Anopheles and Culex larval species. Various chemical parameters of the larval habitat ranging from water pH, temperature, and concentration of ammonia, nitrate and sulphate affect larval development and survival (Mutero, 2004). Mosquito larval control can be possible through manipulations of such parameters (Olayemi et al., 2010). 

The rest measured chemical characteristics such as calcium and magnesium differed significantly across months but had no significant effect on Anopheles and Culex larval densities. The difference of such chemical parameters in larval habitats may be due to the soil particles, chemical characteristics and edaphic factors in the area as mentioned by Soleimani-Ahmadi et al. (2014). It seems further studies are required to investigate the influence of these parameters on development and abundance of Anopheles and Culex mosquito larvae.

All the investigated larval habitats are semi-permanent with still and slightly clear-turbid water in sunlight, making conditions favorable for the development of anopheline mosquitoes rather than Culex. Water of larval habitats at the basement of hills with sandy substrate was found clear during the study period. Turbidity characterized larval habitats with muddy substrate mainly at the beginning of the rainy season.

A limitation of this study that mosquito larvae were not identified at species level. However, previous work carried out in Kassala town confirmed that the major mosquito vectors, Anopheles arabiensis (Hamza et al., 2014), Culex pipiens and Culex quinquefasciatus (Lewis, 1945) were the predominant species; that had been associated with the transmission of infectious agents causing human diseases in the town, hence the findings gained in the current study will be relevant for targeting of vector control measures.

Correlations found between certain chemical parameters and mosquito larval abundance, perhaps, confirms the influence of these parameters on the abundance of Anopheles and Culex mosquito larvae in their breeding habitats. Mosquito larval control can easily be managed through manipulations of chemicals characteristics that affect vector aquatic stages abundance. More studies are needed to fully understand the influence of physicochemical characteristics on the abundance of mosquito larvae in eastern Sudan.

Authors’ contributions

Hamza A.M. carried out the field surveys, data analysis and drafted the manuscript. Saeed K.M. participated in the field surveys and revised the manuscript. Khalid F.A. participated in the field surveys. All authors read and approved the final version of the manuscript.

Acknowledgements

The authors thank Mr. Kowa I (Malaria Center, Kassala State) for his help during the field work. They also thank Mr Ibrahim HE of the Hydrology Unit, Ministry of Irrigation and Water resources, Kassala State, for his assistance in the laboratory work.

Competing interests

Authors declare that they have no competing interests.

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