1 Introduction

Anopheles mosquitoes constitute various species with peculiar behaviour associated with their biting activities and malaria transmission (Oyewole et al., 2007; Nwosu et al., 2011). A large number of Anopheles species have been reported in Nigeria, but the world’s most efficient vectors of human malaria belong to members of Anopheles gambiae sensu lato (s.l.) complex and An. funestus complexes (Oyewole et al., 2007). An. gambiae s.l. is a complex of eight sibling species, which presently includes An. gambiae (sensu stricto) Giles; An. arabiensis Patton; An. bwambae White; An. melas Theobald; An. merus Dönitz; An. quadriannulatus Theobald; An. amharicus Hunt, Coetzee and Fettene and An. comorensis Brunhes, le Goff and Geoffroy (Obembe and Awopetu, 2014). These sibling species are morphologically identical, but genetically distinct. The two members of the An. gambiae complex most responsible for transmission of malaria, An. gambiae s.s. and An. arabiensis, are broadly sympatric, although the latter is more broadly distributed in arid regions (De Souza et al., 2010; Levine et al., 2004). The range and relative abundance of An. arabiensis and An. gambiae s.s. appear to be strongly influenced by climatological factors especially total annual precipitation (Onyabe and Conn, 2001). Tropical areas including Nigeria have the best combination of adequate rainfall, temperature and humidity allowing for breeding and survival of anopheline mosquitoes (Ayanda, 2009). Therefore, malaria could be the largest contributor to total disease burden and productivity losses resulting from major tropical diseases in the country. Demography as well as the socio-economic and cultural characteristic of the population, the predominant vectors and parasites play vital role in the sustenance of malaria burden in the country. In Nigeria, malaria is endemic and stable, being a major cause of morbidity and mortality, resulting in 25% infant and 30% childhood mortality (FMH, 2013). The Roll back malaria (RBM) partnership goal to half the number of malaria infection by 2010 suggests the need for integrated approaches to combat malaria and reduce its consequences.

The composition of mosquito fauna of a pool is influenced by its temporary or permanent nature (Jocque et al., 2010). Rock pools form in eroded depressions on outcropping rock layers and depend on precipitation for filling (Brendonck et al., 2002). The Nigerian northern Guinea savanna is naturally endowed with inselbergs which possess their characteristic flora and fauna (Adebote et al., 2008). Parts of Kaduna State such as Zaria, Kagoro, Kajuru, Nok and Kwoi areas are endowed with inselbergs which possess several of these depressions (NACD, 2013) which rainwater collects to form discrete pools during the rainy season. These collections of water serve as breeding habitats for mosquitoes (Adebote et al., 2008). The species composition, relative abundance and physicochemical factors affecting the breeding of anopheline mosquitoes in rock pool habitats have been the subject of rather little ecological research. Few attempts have also been made to describe the distribution patterns of rock pool biotas in relation to any suspected influence of physicochemical conditions to which the pools are subject and how these in turn affect species composition and relative abundance of preimaginal mosquitoes (Jocque et al., 2010). There are reports on mosquitoes associated with rock pool habitats in northern Nigeria (Adebote et al., 2008; Service, 1974). This article presents the results of species composition, relative abundance and physicochemical conditions of breeding microhabitats of Anopheles mosquitoes exclusively in rock pools. A detailed knowledge and understanding of the malaria vector species' composition, abundance, and physicochemical factors influencing their breeding is therefore relevant in understanding their role in malaria transmission and hence its control.

2 Materials and Methods

2.1 Study area and sampling sites

This study was carried out on the exotic plateaux and rocky hills of Kaduna State located between Lat 8°30'0''N and 11°30'0''N and Long 6°0'0''E and 8°30'0''E, Northern Nigeria. Vegetation type comprises tropical grassland in the southern Guinea Savannah to Sudan Savannah (NACD, 2013). Rainfall is heavy in the southern part and around Zaria in the northern part; with average rainfall of about 1016mm (NACD, 2013). The State enjoys a rainy season of 5 months in average. Available rock pools sampled for mosquito breeding were spatially distributed in 21 settlements endowed with inselbergs. Sampling locations were randomly selected within the three Geopolitical zones of the State. Seven Local Government Areas out of the 23 in the State were sampled according to savannah areas. The southern Guinea Savannah area comprise: Jaba, Jema’a, Kachia, Kagarko, Kaura, Sanga and Zango-Kataf, Birnin-Gwari (partly Guinea Savanna and partly Sudan Savannah), Chikun, Igabi, Lere, Kaduna- North, Kaduna-South, Kajuru, Kauru, whilst the Sudan Savannah areas are: Kubau, Kudan, Makarfi, Ikara, Giwa, Zaria, Sabon-gari and Soba. Jere, Kagarko (Kagarko L.G.A.), Kwoi, Nok, Chori, Samban-Gida (Jaba L.G.A.) and Kagoma (Jema’a L.G.A.), Kangimi (Igabi L.G.A.), Malali (Kaduna-North L.G.A.), Baban-Sora, Kujama (Kajuru L.G.A.), Kajuru, Tudun-Mare and Kufana (Kajuru L.G.A.) settlements were sampled in Guinea Savannah. Hanwa (Sabon-Gari L.G.A.), Danmagaji, Wusasa, Kufena, Dutsen-Abba, Dumbi and Zango-Aya (all in Zaria L.G.A) settlements were sampled in Sudan Savannah.

2.2 Determination of geographic attributes

The study was conducted for 5 months between June and October, 2013. Available true rock pools (as opposed to edged rock pools) were searched fortnightly on the inselbergs as much as access permitted, assisted by the local community members. A GARMIN e Trex Venture HC hand held global positioning system (GPS) was used to determine the geographic coordinates, altitude above sea level and altitude above the surroundings of the rock pools in the settlements across the State.

2.3 Determination of physicochemical parameters of rock pools

Mean depths of water in rock pools were determined by dipping a metal rod to touch the bottom of the pools at 3 locations and wet regions measured against transparent ruler. The surface areas of rock pools were determined from length and width measurements with a meter rule, while making necessary adjustments to irregularly shaped surfaces. The pH, electrical conductivity, total dissolved solids and temperature of water in each rock pool were determined by means of a HANNA HI 98129 pH/ EC/TDS/Temp meter (Adebote et al., 2008). Other parameters which include turbidity, total alkalinity, total suspended solids, total hardness, chemical oxygen demand, phosphate, and nitrate were determined according to standard protocols described by APHA (1998).

2.4 Sampling and specimen preservation techniques

Samplings were carried out forth nightly in the 3 senatorial zones of the State for a period of 5 months. Point distances between pools were determined with tape. Ten dips of the water in every other rock pool were obtained with a plastic soup ladle dipper whose capacity is 0.105 L (Service, 1976). The water were collected in a white plastic bowl and carefully observed for the presence of preimaginal mosquitoes. Anopheline larvae were collected alive into plastic bottles, transported to the laboratory and reared to adults in small plastic bowls (11 x 5.5 cm) on a diet of bakers’ yeast (Service, 1993). Adult anopheline mosquitoes were preserved dried on silica gel in labeled specimen bottles.

2.5 Species identification

Anopheline mosquito’s identification was based on the pictorial keys of Gillies and DeMeillon (1968) and Gillies and Coetzee (1987). For specimens of Anopheles gambiae s.l., genomic DNA was extracted using the method of Collins et al. (1987). Polymerase Chain Reaction (PCR) based on species-specific single nucleotide polymorphisms (SNPs) in the intergenic spacer region (IGS) were used for the identification of member species of Anopheles gambiae complex. To each of 12.5 µl reaction, 1µl of primers (GA, AA, ME, QD and UN) were used. Other reagents used for isolation of DNA include PCR Buffer x10, dNTP₃, MgCl₂, Water and Rtaq. Tris-buffer and Sodium Diodecyle Sulphate (SDS) were added to maintain and lyse the DNA respectively. Samples were run for 95°C/5mins for 1 cycle; 95°C/30sec, 50°C/30sec, 72°C/30secs for 30 cycles and another 72°C/5mins for 1 cycle. Amplified fragments was analyzed by electrophoresis in a 1.5% agarose gel with 14µl of ethidium bromide and visualized under ultraviolet light Illuminator (Scott et al., 1993).

2.6 Statistical analysis

Relationship of physicochemical parameters of sampled water with mosquito species’ abundance was sought in a correlation assay (Pearson’s correlation). Principal component analysis (PCA) was used to examine the interaction effects among physicochemical parameters of water and to select water parameters that were most useful in distinguishing mosquito species habitats in rock pools. One-way analysis of variance (ANOVA) was employed to analyze data on response variables against physicochemical factors in relation to mosquito abundance (SPSS version 17.0). Least significant difference (LSD) was used to separate significantly differed means. All levels of statistical significance were determined at α = 0.05.

3 Results

The relative abundance of mosquito species in the study area was expressed as the corresponding percentage of the total number of Anopheles collected (Table 1). Inselbergs at Zango-Aya and Hanwa recorded higher number of pools (25 each) while Wusasa recorded lesser pools (6). Kufana inselbergs were thoroughly observed without any breeding microhabitats supportive of anopheline mosquitoes. The actual height of the inselbergs was determined as the resultant of the height above sea level and above the surroundings. A total of 12 (57.14%) of the 21 inselbergs supported anopheline mosquitoes' breeding from which 84 adults were reared successfully. Identified mosquitoes belonged to 5 species and 1 species complex which included 2 (2.38%) An. arabiensis Patton, 45 (53.77%) An. gambiae Giles s.l., 30(35.71%) An. gambiae Giles s.s., 1 (1.19%) An. longipalpis Theobald, 1 (1.19%) An. pretoriensis Theobald and 5(5.95%) An. rufipes Gough. The relatively higher percent of one of the most efficient and dominant vectors of malaria occurred on Dumbi (36.90%) and Kangimi (17.86%) inselbergs (Figure 1).

Table 1 Species abundance of Anopheles mosquitoes breeding in rock pools on inselbergs in Kaduna State, Nigeria

A total of 77 Anopheles gambiae complexes bred in rock pools on inselbergs in Kaduna State (Table 2). Geopolitically, the number found decreased in trend from northern, central and southern (39, 21 and 17) parts respectively. The Anopheles gambiae species specific PCR revealed 41.6% amplification. 38.9% population belongs to An .gambiae s.s. with band range of 390bp whilst 2.6% were An. arabiensis with band range of 315bp. An. gambiae s.s. was predominant in all the collections with two sibling species occurring within the northern Guinea savanna. 58.4% of the An. gambiae complex could not be identified through PCR even after 3 runs. This could probably be due to popping open of the PCR tubes during its cycles which left the popped open tubes dried. The samples could also not properly mix with primers. The 2.6% were recorded in rock pools in central and northern parts of Kaduna State.

Anopheline mosquitoes bred in pools with large surface areas in relatively shallow depths (Table 3). Temperature of the microhabitats ranged from 22.8 to 36.3°C; the high extremes were associated with An. gambiae s.l. and Anopheles rufipes. The anopheline species were associated with pools of alkaline nature (pH 8.89–12.12). Total dissolved solid (TDS) and electrical conductivity (EC) obtained in this study were largely below detectable limit with HANNA HI 98129 pH/ EC/TDS/Temp meter. Ranges of hydro-physical (depths; p-value = 6.9x10^-11 and surface areas; p-value = 9.9x10^-11) differ significantly (P < 0.05); but those of hydro-chemical (pH, TDS, TSS, turbidity, hardness, COD, PO₄, NO₃, alkalinity) parameters did not differ significantly (P > 0.05) amongst species. Principal component analysis (PCA) showed that pH had positive correlation amongst the rock pool habitats of mosquitoes while total suspended solid (TSS) tended to be neutral. Nitrate, phosphate, alkalinity, turbidity, total dissolved solid, electrical conductivity and chemical oxygen demand (COD) had negative interactions amongst the rock pools. Water temperature and hardness had slight positive interactions, thus vital in determining spatial distribution of anophelines in rock pool habitats. ND, not determined, fall within the ranges of pool habitats that were not tested for hydro-chemical properties.

4 Discussion

Results of this study revealed that discrete microhabitats supportive of anopheline mosquitoes existed in rock pools on inselbergs and each pool constituted an independent replicate for determining their ecology. This was a significant observation. However, observed vector species composition and related malaria transmission is known to be influenced by the environmental changes and changes in human practices (Sinka et al., 2010). This supported several unique features of the inselbergs observed in this study which include the inevitable presence of humans on the inselbergs and close proximity of the inselbergs to human habitation. Children and adolescent utilized the inselbergs for adventure. Parents are either consistently quarrying on the rock or utilizing the rock as platform for drying their harvested farm produce. Churches of different denominations have acquired most of the inselbergs as outstations and camping grounds for special prayers throughout the seasons. Therefore, considering the anthropophilic nature of Anopheles gambiae s.s. and exophilic nature of An. arabiensis (Sinka et al., 2010) which are well adapted to take advantage of temporary aquatic habitat associated with human activities (such as farming, construction and quarrying), these vectors undoubtedly have contributed substantially to malaria burden within the sampled localities. In terms of chances of malaria transmission, the present study contradicts the findings of Adebote et al. (2008) which showed that rock pools do not constitute natural breeding habitats for malaria vectors around Zaria, Nigeria. In this study, it was revealed that Dumbi and Kangimi inselbergs recorded an overall dominance of An. gambiae s.l. and An. gambiae s.s. in rock pools. Collins and Besansky (1994) showed that these malaria vectors have marked preference for human environments and adapt so rapidly to changes in the environment induced by human habitation and agriculture. Human habitations, abundance of rice fields which constitute the most productive habitat for An. gambiae s.l. (Gillies and De Meillon, 1968; Mwangangi, 2006) and quarrying activities obviously account for its attendant consequence in larval habitat disturbance that favours the colonization of rock pools by the most efficient vectors of malaria. Rock pools at Dumbi, Kufena, Jere inselbergs were excavated to increase their water storage capacity which was manually used by the residents to irrigate their crops due to rain seizure. Rock pools at Kufana were supposedly dried but at the particular time of sampling the pools were filled with fresh rainfall. The pools were sampled for bio-organisms and physicochemical parameters without encountering any mosquito larva.

The moderately high proportion (38.9%) of An. gambiae s.s. in the northern Guinea savannah contrasted other reports that highlighted widespread occurrence of An. gambiae s.s., the major malaria vector in Nigeria (Oyewole et al., 2005). Anopheles arabiensis is considered a species of dry savannah environments and sparse woodland, yet it is known to occur in forested areas, but only where there is a history of recent land disturbance or clearance (Harbach, 2004). The lower proportions (2.6%) of An. arabiensis were recorded in northern and central parts of Kaduna State which have least amount of rainfall seasonally compared to the southern parts. In Nigeria, higher populations of An. arabiensis were recorded in the arid northeast region, while higher populations of An. gambiae s.s. were collected in the southern tropical rainforest (Awolola et al., 2003; Oyewole et al., 2005). Onyabe and Conn (2001) recorded An. arabiensis in northern Guinea savannah, Nigeria. Previous study by Kenea et al. (2011) reported the abundance of larvae of the principal malaria vector (An. arabiensis) in sand pools along the edge of the Meki River during the dry and small rainy season in Ethiopia. The 3 anophelines (An. longipalpis, An. pretoriensis and An. rufipes) are generally man-bitters and are regarded as incidental vectors of human, thus doubtful of their public health importance (Gillies and De Meillon, 1968).

Prevailing physicochemical parameters of the rock pools are important factors for survival and development of mosquito. This study observed an overall wide temperature range slightly above the recommended optimum temperature (31°C) considered as the most rapid for larval and pupal development (Oyewole et al., 2009). The apparent rise in optimum temperature of the rock pools could be attributed to abrupt rise of the inselberg above the ground positioning the pools to direct incident radiation from the sun. Jocque et al. (2010) observed that water temperature in rock pools depends on climate and seldom exceeds 40°C because of the balance between cooling through evaporation and heating by insolation. The relatively shallow depth of the rock pool habitats influences the species abundance of anopheline mosquitoes. This natural selective pressure controls the immigration of its predators into such pools that appear to be somewhat constrained to deeper pools. Habitat size also influenced the species composition of mosquitoes in rock pools. Rock pool habitats with larger surface areas were observed to be densely inhabited with tadpoles of both frogs and toads which are natural enemies of mosquito larvae and this possibly resulted in decreased species composition. Habitats that are larger in area tend to have higher immigration and often offer a wider range of microhabitats/niches and decreased local extinction, which result in higher species diversity (Kumar and Hwang, 2006). Therefore, it is not surprising that observed rock pool habitats that had higher immigration of amphibian recorded low mosquito species composition in this study.

This study affirmed that alkaline media is binding on the relative abundance of anopheline larvae, and thus played pivotal role for their prolific breeding in rock pool habitats. Increased surface pH in water bodies is due to increased metabolic activities of autotrophs, because in general they utilize the CO₂ and liberate O₂ thus reducing H⁺ ion concentration (Bellingham, 2013). Rock pools observed in this study were largely devoid of organic nutrients and this invariably decreased the pH of the breeding microhabitats due to carbonate chemistry (Bellingham, 2013). Findings on mosquito species abundance and its relation with pH did not corroborate with reports of CDC (2004) that mosquito larvae are distributed in natural waters of varying pH. In this study, acidic environment was not implicated; however, this is not sacrosanct given impacting influence of organic nutrients (Bellingham, 2013). The analysis of results on electrical conductivity and total dissolved solid agrees with the work of Ranta (1982) that the small nature of the volume of water in rock pools results in strong fluctuation in electrical conductivity. Jocque et al. (2010) observed that rock pools are characterized by low conductivity immediately after filling, typically fluctuating between 10 and 30 µS cm^-1. As the water evaporates, conductivity increases mainly because of the concentration of metabolites and can reach values up to1400 µS cm^-1 in pools with the longest hydroperiod. Inorganic nutrients such as nitrate, phosphorus, hardness, total suspended solid, alkalinity, chemical oxygen demand, and turbidity had no significant effect on the abundance of mosquito larvae breeding in rock pools. This is in agreement with Jocque et al. (2010) that nutrients such as nitrate, phosphorus, alkalinity decline quickly because of nutrient uptake by organisms and a reduced rate of nutrient supply from the sediment. Immediately after filling, dissolved nitrogen and phosphorus concentrations may be quite high but nutrients in the sediment get into the water via bioturbation by tadpoles and some crustaceans. Published data on nutrient availability on inselbergs indicate that phosphorus and nitrogen are severely limited (Dörrstock et al., 1996). Removal of nutrients from the system is largely by flushing by intense rain, sediment erosion by wind and, to a lesser extent, by terrestrial predators and scavengers removing organisms from the pool basins, and possibly also by the emergence of adult insects (Jocque et al., 2010).

Epidemiologically, encountered mosquito species were potential vectors of malaria and filariasis. Two important malaria vectors (Anopheles gambiae s.s. and An. arabiensis) were genomically identified breeding in sympatry in rock pool habitats within Kaduna, Nigeria (perhaps, for the first time). Water pH and temperature appeared to play significant determinant roles in the occurrence of preimaginal mosquito species in rock pool habitats. Use of mosquito control tools that will not ruin or harm biological control prospects but which incorporate indigenous community participation should be contemplated in rock pools to stem vectorial roles of identified species.

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