1 Introduction

Malaria has remained a leading public health problem in Nigeria with 97% of the population at risk. According to (NMCP, 2014), malaria accounts for about 60% of outpatient visits and 30% of hospitalizations in Nigeria. It is a leading cause of mortality in children under five years of age and is responsible for an estimated 225,000 deaths annually. It also contributes to an estimated 11% of maternal mortality and 10% of low birth weight. Malaria is transmitted throughout Nigeria and five ecological zones define the intensity, seasonality of transmission and mosquito vector species: mangrove swamps; rain forest; Guinea - savannah; Sudan - savannah; and Sahel - savannah. The duration of the transmission season decreases from year - round transmission in the south to three months or less transmission in the north. Nigeria, with a population of about 172 million reports more death due to malaria than any country in the world. Thus, the country contributes one fourth of the world’s malaria (Nigeria, 2010).

Of the 37 Anopheles species documented vectors of malaria in Nigeria, Anopheles (An.) gambiae s.l. and An. funestus group are the major vectors (Afolabi et al., 2006). Within the An. gambiae complex, An. arabiensis predominates in the north and An. melas in the mangrove coastal zones. Other localized vectors include An.nili, An.coustani, An.moucheti and An. squamosus.

Enugu State, Nigeria has an average malaria incidence rate of 15% (MVCU, 2000). The stability of malaria in the area is generally due to highly efficient vectors and multiple primary vector species that are present throughout the year. Likewise, climatic factors have direct impact on the entomological indices involved in malaria transmission as this was observed in the studies of (Zyzak et al., 2002; Uttah and Uttah, 2013).

Microclimatic variation has all-around effects on malaria transmission in the African highlands where temperature, humidity and rainfall affect the abundance of mosquitoes. Changes in the microclimate of an area may make the areas previously too cool for vector population establishment now suitable, causing an expansion of vector species to vast area leading to an increase in chance of disease transmission. Synergistic effects between temperature and rainfall on vector ecology may produce malaria transmission. Increase in temperature may facilitate larval development, enhance vector survivorship and reproductive fitness, and increase the blood feeding frequency and parasite sporogonic development rate in the previously cooler areas. These effects have been explained using scenarios from anthropogenic environmental changes in east Africa and other parts of the world. In East African Highlands, (Zhou et al., 2004) reported that a 1°C increase in minimum temperature with a lag of time of 1-2 months and 1°C increase in maximum temperature with a lag time of 2-5 months led to an 80-95% increase in the number of malaria outpatients. In the highlands of Uganda, (Lindblade et al., 2000) compared mosquito density, biting rates, sporozoite rates, and entomological inoculation rates between eight villages located along natural papyrus swamps and eight villages located along swamps that have been drained and cultivated. They found that on average all malaria indices were higher near cultivated swamps. Maximum and minimum temperatures were significantly higher in communities bordering cultivated swamps. The average minimum temperature of a village was significantly associated with the increased number of Anopheles gambiae per house. Thus, replacement of natural swamp vegetation with agricultural crops led to increased temperatures and elevated malaria transmission risk in cultivated areas.

Simon-Oke et al. (2012) in his study in Nigeria demonstrated that temperature between 26°C and 32°C with average humidity of 55% facilitated the higher mosquito abundance. Similar studies have also been previously reported on the maximum survival rate of mosquito for the related temperature and humidity (Murty et al., 2010).

Therefore meteorological factors are important drives of malaria transmission. Ambient Temperature plays a major role in the life cycle of the malaria vector. Temperature between 15°C - 40°C and humidity between 55% and 80% are suitable for the completion of the Plasmodium falciparum and Plasmodium vivax malaria parasites life cycle in the mosquito (Zhou et al., 2004). The sporogonic cycle takes about 9-10 days at temperature of 28°C but stops at temperature below 16°C (Lindsay and Birley, 1996). The daily survival of vector is dependent on temperature as well. At temperature between 16°C and 36°C, the daily survival sustains but drops rapidly at temperature above 36°C. The highest proportion of vector surviving incubation period is observed at temperature between 28°C and 32°C (Craig et al., 1999).The gonotrophic cycle which is the time between blood meals of the vector is short at higher temperatures because digestion speed increases (Haque et al., 2010). Therefore higher temperature results in more frequent vector – host contact.

Thu et al. (1998), also submitted in his study that humidity is one of the vital factors affecting the life cycle pattern of mosquitoes and it has been observed that the temperature at 28°C with 50 – 55% relative humidity is the most appropriate condition for the elevation in mosquito density or abundance than the condition of lower temperature with higher humidity (22°C /80 – 85% RH).

Rainfall provides breeding sites for mosquito to lay their eggs and ensures a suitable relative humidity of at least 50-60% to prolong mosquito survival. Relative humidity below 60% shortens the life span of the mosquito vectors (Rogers and Randolph, 2006). In the study of (Simon-Oke et al., 2012), they suggested the temporal change in mosquito abundance is mainly caused by rainfall. An. gambiae adults are more abundant during the rainy season than during the dry season. Regular data collection and analysis of entomological parameters; such as species composition and their abundance in an area, feeding and resting behaviours linked to the data collected on climatic, epidemiological and environmental factors serve as the most appropriate interventions as well as when and where to apply them (WHO, 2013). The inter-relation between the malaria vectors, man and Plasmodium species is also influenced by climatic factors (temperature, relative humidity and rainfall), seasonal fluctuation of the vectors, abundance and distribution of the mosquito species. (Alten et al., 2000) opined that exact information on the seasonal prevalence of mosquito fauna in a region is essential for the development of efficient vector control programs. However, information on the seasonal variation, hourly biting rhythms of Anopheles mosquito and the impact of microclimatic variables on these Anopheles species in the south-east Nigeria is however scant.

We undertook this study with two main objectives of ascertaining the abundance of malaria vector and to determine the relationship between the micro-climatic factors, seasonality and the vectors behaviour on malaria transmission in Ahani- Achi.

2 Materials and Methods

2.1 Study area

The study was undertaken in Ahani-Achi community (06^°37^’N, 07^°52^’E) in Oji River Local Government Area of Enugu State, Nigeria. Ahani-Achi as a community has four villages namely: Oruchi, Mgbaragu, Okpuno, Umuelewe. The community has an estimated population of 5,000 people (NPC, 2006) comprising mainly farmers, civil servants, petty business men and women. There are two health facilities in the community which includes a Primary Health Care centre and a private clinic. The community is characterized by wet (April to October) and dry season (November to March). The annual mean rainfall of 1 800 mm and the mean monthly temperature varies between 23°C and 31°C. The known river “Oji River” traverses the villages in the community. Insecticide treated net distribution has been carried out in the area.

2.2 Mosquito collection

A modified WHO light trap and Pyrethrum Spray Catch technique were used in collecting outdoor and indoor human biting & resting mosquitoes. Suitable house structures were randomly selected based on the number of children and pregnant women in the house, houses with an average number of four people, acceptability and well ceiled structures were selected. Based on these criteria, eight houses were selected for WHO light trap and twenty four houses for PSC in the study area.

Collection with the light trap was done both indoor and outdoor all through the night from 18:00 to 06:00 GMT bimonthly with the collection cups changed hourly to evaluate the vector behaviour. An untreated bed-net was provided to be used during the night that the light traps were used in the house both indoor and outdoor (Mboera et al., 1998). The light traps were hung approximately 1.5 meters above the floor. The Anopheles species abundance both indoors and outdoors with the time of collection were determined and recorded throughout the whole sampling period in the study site. Thermohygrometer was used to monitor the Temperature (°C) and Relative Humidity (%) changes.

Indoor resting mosquitoes at the study site were collected using PSC technique. The technique was carried out in all the twenty four selected houses in Ahani Achi, six per village. The collections were conducted from 6.00 - 9.00 GMT. We made sure all the movable properties were taken outside the sprayed room. Food and food stuff were first taken out of the room. White sheet were spread out on the floor of the room for the knocked down mosquitoes and other insects to fall into it. Anopheles species collected from the sprayed rooms were examined according to the external appearance of the stomach contents using the dissecting microscope as described by (GMEM, 1994). The female mosquitoes were classified based on their abdominal condition as unfed, fed, half gravid and gravid. The degree of coiling of ovarian tracheoles was then observed to determine if the female is parous or nulliparous as to determine their age. Morphological identification was done using the keys of (Gillies and De Meillon, 1968; Gillies and Coetzee, 1987). Mosquito collection using both the light trap and PSC were carried out bimonthly.

2.3 Entomological indices

The Indoor resting density and Man-biting rates were measured from the result of the PSC collection. The density of the Anopheles species collected indoor using the PSC were measured by the number of Anopheles mosquito collected divided by the total number of house sampled and total number of night the collections were made.

Indoor Density (D) = (number of females ÷ number of houses) ÷ number of nights

Man-biting rate is expressed as the number of bites a person receives from a specific vector species per night. In this study, this index was calculated from the PSC collections as the total number of freshly fed females of a species divided by the total number of occupants who spent the night in the rooms and then the total number of nights that were used for the collection.

Man-biting rate (Mbr) = (number of freshly fed females ÷ total number of occupants) ÷ total number of nights

2.4 Data analysis

Total mosquito catches, species collected, season of collection and environmental variables (temperature and humidity) were recorded. The relative frequency of each species was calculated against the total catch during the sampling period. Relative abundance of members of the Anopheles species was determined against each collecting technique. Resting density of the Anopheles mosquitoes collected indoors using PSC was calculated as the number of female Anopheles mosquitoes per number of room per number of nights. Man-biting rate expressed as the number of bites a person receives from a vector species per night was calculated indirectly as the total number of freshly fed female Anopheles collected each day divided by the total number of occupants who slept in the room the previous night before the collection was made. Univariate and multivariate analyses were performed. Seasonal variation in mosquito catches was analysed using ANOVA. Multiple regression analysis was used to explain the variation in total mosquito catches with respect to the following micro-climatic variables (temperature and humidity). Pearson correlation analysis was then used to assess the relationship between collections and the micro-climatic variables. All statistical analyses were performed at 5% significance and comparison between collections point (indoor and outdoor) was only done during the months when collections were done.

Verbal consent was sought from the community head and all the household head for this study. Owners of the selected households and district authorities were sensitized prior to the study and their permission obtained, while the privacy of the individual participants and household members were highly protected. Collectors were selected from the community and the health centre to facilitate acceptance from people. Informed consent was obtained from each collector.

The collectors were trained on how to collect the mosquitoes that were trapped in the light traps and change the collection cups hourly. They were also trained to assist in collecting pyrethrum knocked down mosquitoes. Bed nets (LLINs) were given to each of them and the participating household in the study.

2.5 Limitations of the study

All efforts to get accurate data from Nigerian Metrological Agency (NIMET) on rainfall proved unsuccessful as there was no place to get a rain-guage for the daily rainfall reading especially during the rainy season.

3 Results

3.1 Species composition

A total of 1570 Anopheles mosquitoes representing 4 species groups were collected in the study area during April, 2013 to February, 2014. Of the total Anopheles species collected, 911 were collected using WHO light traps and 659 using the PSC technique. Among the 4 species groups collected, An. gambiae sl (n = 1170, 74.5%), An. funestus group (n = 294, 18.7%), An. nili complex (n = 59, 3.76%) and An. coustani complex (n = 47, 3.0%). All the 4 species groups (An. gambiae sl, An. funestus group, An. nili complex and An. coustani complex) were collected using the light trap while 3 of the species (An. gambiae sl, An. funestus group and An. nili complex) were collected using the PSC technique.

3.2 Seasonal abundance of female Anopheles mosquitoes

The rainy season in the study area spans from April through October. For equality in the number of months making up the rainy season and dry months, we divided the seasons into early rain (April to July), late rain (August to October) and dry season (November to February). Using the light trap (Table 1), more collections (n = 414, 45.44%) were made in the early rainy season (April- July), followed by the late rainy season (n = 349, 38.31%) and the least collections (n= 148, 16.25%) made in the dry season (November- February).

Total collections made indoors were higher (n = 507, 55.65%) than the collections made outdoors (n = 404, 44.35%), though there is no significant difference (P = 0.456, P >0.05) in the abundance of the malaria vector indoor and outdoor. Of the species collected indoor and outdoor using the light trap, An. gambiae sl were more abundant (n = 635, 69.70%) followed by An.funestus group (n = 180, 19.76%), An nili sl (n = 49, 5.38%) and An. coustani complex (n = 47, 5.16%).

3.3 Hourly distribution of mosquito species collected during the study period

The hourly distribution of mosquitoes (Figure 1) collected for the various time groups for both indoor and outdoor collections were pooled together. The hourly distribution of mosquitoes sampled from the field started from 6 pm to 6 am. There was an increase in hourly activity from the starting hour (6 pm), with peak of activities between the hours of 2 am -3 am (Indoor) and 3 am -4 am (Outdoor), then eventually declining till 6 am.

3.4 Climatic variables affecting the abundance of malaria vectors in the study area

During each collection period, microclimatic variables (temperature & relative humidity) were measured using thermo-hygrometer. Figure 2 and Figure 3 below were obtained from a pooled monthly temperature and R.H readings collected from the structures where the light traps were used in sampling both endophagic and exophagic malaria vectors. These chats indicate the response of vectors to atmospheric temperature and relative humidity in their feeding activities.

3.5 Indoor biting and resting Anopheline collections

A total of 659 Anopheline mosquitoes were collected using the PSC. The species comprises An. gambiae s.l (n =535, 81.18%), An. funestus group (n =114, 17.30%) and An.nili complex (n =10, 1.52%). Twenty four households were sampled in the community within the study period. Table 2 shows the seasonal collections of Indoor biting Anopheles species using PSC.

From the above Table 2, the collections made during the late rainy season (n =317, 48.10%) were more than that of the early rainy season (n =250, 37.94%) and dry season (n =92, 13.96%). Seasonally, An. gambiae s.l were more dominant in the collection (n =535, 81.18%) than An. funestus group (n =114, 17.30%) and An. nili complex (n =92, 1.52%). An.funestus group were more (n =61, 53.51%) in the early rainy season than the dry season (n = 44, 38.60%) and the late rainy season (n =9, 7.90%).

Abdominal conditions of the Anopheles mosquitoes collected with PSC were assessed. Based on abdominal conditions, they were grouped as: unfed, fed, half gravid and gravid. Unfed females has a dark and flattened abdomen; fed had a dark red abdomen with blood occupying most of the abdomen; in half gravid, blood occupied only 3–4 segments of the ventral surface and 6–7 segments of the dorsal surface of the abdomen; in gravid females, most blood was digested and the abdomen was whitish and distended. The chat in Figure 4 showed that of the 659 Anopheles species collected, (n = 488, 74.05%) were fed, (n = 110, 16.7 %) were half-gravid, (n = 47, 7.13%) gravid and (n= 14, 2.12%) were unfed.

Indoor resting density (IRD) and Man biting rate (mbr) which is indices of actual incidence of man- vector contact were estimated from the PSC collections (Table 3).

The monthly collection showed that more collections were made in the months of September (n =114) and October (n =112) while least collection in the month of January (n =12) followed by the month of February (n =17). The monthly indoor resting density of the anophelines shows that An. gambiae sl are likely more abundant indoors with 66% probability, An. funestus group with 25% probability and An.nili complex with 1% probability. The man-biting rate of the different species collected in the study area also showed that An. gambiae sl had the highest biting rate indoors with 3.9 bites / man / night, 1.5 bites / man / night for An. funestus and 0.1 bite /man/ night for An.nili.

In all the collections using the PSC, parous rate was 61.3%, and this ranged from 54% in April – July, 38% in August – October and 92% in November 2014 -. February 2015 (Table 4). The variability in parity rates between the seasons was statistically significant (P <0.05).

4 Discussions

In the study, four species of Anopheles were collected namely: An. gambiae s.l, An. funestus group, An. nili complex and An. coustani sl. These species have been reported to be in close association with human (Russell et al., 2011). Though a very large number of Anopheles species have been reported in Nigeria (Oyewole et al., 2005; Molineaux and Gramiccia, 1980) but the main vectors of malaria as reported by (Service, 1970; Ukpai and Ajoku, 2001) belongs to the members of Anopheles gambiae s.l and An. funestus group and these were the major species collected from the study. Secondary malaria vectors like An. coustani complex and An. nili complex collected in the study have also been reported in the country by (Gillies and De Meillon, 1968; Gillies and Coetzee, 1987). The mosquitoes collected in the study proved that most anthropophilic anophelines feeds at night when the victims are resting indoors or outdoors after the daily activities and this is in line with the study of (Moreno et al., 2007; Hay et al., 2011) where the vector involved in transmission of malaria were abundant and exhibited nocturnal anthropophily.

The studies of (Toure et al., 1994; Fontenille et al., 1997) suggested that the temporal alteration in Anopheles mosquito abundance is mainly caused by seasonal fluctuation (from wet- dry season) as Anopheles mosquitoes collected in this study were more abundant during the rainy season than during the dry season. And this is consistent with the findings of (Zhou et al., 2007). Using the both collection techniques (PSC and light trap), 42.3% of the malaria vectors were collected in the early rainy months, 42.4% in the late rainy months while 15.3% were collected in the dry months Anopheles gambiae s.l were collected more in the late rainy season and least in the dry season. Increase in the abundance of vectors in the rainy season suggests an increase in the temporary breeding sites within the study area as observed in the study of (Echodu et al., 2010). An. funestus and An. coustani were more abundant during the dry season and the study of (Mwangangi et al., 2013) proved that An. funestus and An. coustani have the tendency to survive in little available breeding habitats with minimal water collection after rainy season aside breeding in large non - flowing water bodies during the dry seasons.

The aim of collecting Anopheles species indoors and outdoors was to assess the feeding behaviour of the vectors. Collection of malaria vectors with different techniques have provided information on their biting behaviour, composition of the Anopheles fauna during periods of high mosquito densities and their resting preference (Aigbodion and Nnoka, 2008; Duo-quan et al., 2012). Collections with the light traps showed that more collections were made indoors (55.65%) than outdoors (44.35%), though there was (P =0.456>0.05) no significant difference in the abundance of the malaria vector indoor and outdoor. Pyrethrum Spray Catch collection showed that An. gambiae and An. funestus were the most indoor biting and resting Anopheles mosquito in the study area. Oyewole et al. (2007) reported that An. gambiae and An .funestus were largely responsible for the indoor biting activity but the former was found to be highly endophilous compared to other species. This complies with our finding that the species preferred to rest indoors after feeding and similar result was recorded in behaviour was recorded in the study of (Mwangangi et al., 2013), but with lesser population An. funestus indoors. Anopheles nili complex has been described as an efficient malaria vector (Fontenille and Simard, 2004). Anopheles nili is usually responsible for a high nuisance to humans in villages along rivers, and abundance rapidly decreases within a few kilometers from the breeding sites (Le Goff et al., 1997) as this paints the picture of the distance from the river “Oji-River” axis to Ahani- Achi community. It is also present at the periphery of urban areas. In this study, they were collected both indoors and outdoors confirming the findings of (Awono-Ambene et al., 2009; Antonio-Nkondjio et al., 2009). An. coustani was not collected using the PSC technique, as this substantiates the findings of (Mwangangi et al., 2013) who implicated this species for outdoor malaria transmission where the exposure level to Plasmodium falciparium was higher outdoors than indoors.

Biting activity of the anophelines in the study area commences effectively at the early part of the night and peaks at 2-4 am for both indoors and outdoors when the inhabitants are sleeping. The extensive biting activity of An. gambiae and An. funestus into the latter part of the night could be confronted by the use of insecticide-treated bed-nets, wearing protective clothes and using repellents. Also the people’s lateness to bed (mostly 11 pm –12 am), the non-compliance to the use the bed-nets and poor conditions of the nets due to the duration of usage (for those using the bed-nets) are implicated to have modified the known biting peak of 12-1 am, (22) to 2 am-3 am (indoors) and 3 am-4 am (outdoors). This finding tally with that of (Atangana et al., 2009) in which the change in feeding behavior as seen in this study is associated with availability of people and seasonality (people tend to be indoors by that time either because of rain or the cold weather).

The room density of the mosquitoes caught indoors was approximately one Anopheles mosquito per room per night, with a probability of 66% chance of the species being An. gambiae sl and 25% probability of the Anopheles species being An. funestus. The probability of having An. nili biting and resting indoors in Ahani Achi was as low as 2% per room/night. The finding on mosquito density per room suggests that approximately one Anopheles species may feed on each occupant of the house per night as the houses selected were based on the average of 4 occupants in a room. This is of serious public health attention because An. gambiae sl and An. funestus complex are very efficient malaria transmitters and can infect many people in a place even at lower densities.

A biting rate of 5.5 bites / man / night were recorded indoors of which 3.9 bites/man/night were for An. gambiae, 1.5 bites / man / night for An. funestus and 0.1 bite /man/ night for An. nili. These biting rates are enough to intensify the spread of the infection in the study community and can jeopardize the malaria elimination efforts of the ministry of health.

Parity rate determination of the indoor resting mosquitoes collected indicated 61.3% of the female Anopheles species were parous. The parity rate observed in the study showed that female Anopheles species collected during the dry season were more parous (92%) than those collected during the late rainy season (38%) indicating that the eggs and larvae being washed off by the heavy rains. The result of parity rate in this study is in agreement with the study of (Uttah et al., 2013b) where the parity rate in the dry season was higher than that of the rainy season. This abundance of parous female mosquitoes during the dry season months could not be due to higher mortality during the rainy season because the higher humidity level during this season provides favorable conditions for survival. The result in higher percentage of nulliparous mosquitoes during the rainy season was due to the availability of more breeding sites. As the dry season gets in progress, some of the breeding sites dry up completely while the permanent ones thin out both in area and yield. This effectively translates to a drop in the number of younger females into the female population thereby increasing the proportion of parous ones.

Though (Paaijmans et al., 2010) affirmed that temperature is directly affecting mosquito breeding, survival, and behavior and also malaria transmission but we were not able to find any significant (P =0.456>0.05) association with temperature and Relative humidity {(r = -0.216,P= 0.456>0.05) (r= -0.595(indoor), -0.477 (outdoor)} with abundance of female Anopheles mosquitoes in the study using the spearman’s rho correlation test. Bashar and Tuno (2014) also reported not finding any significant association with temperature; mosquito density and malaria transmission in their study. From the study relative humidity was observed to be above 60% and it increased from 18.00 hr – 6.00 hr which was the time of the collection. Increase in the relative humidity at night gives an insight into why the vectors’ biting activities are predominantly at night because the R.H at night is favorable for the mosquitoes to thrive on. The reason for not having any significant relationship with temperature and R.H in this region might be because the microclimatic factors are always suitable for mosquito breeding, development and feeding. Moreover, statistical significance alone does not always unlock the complex biological dynamics of mosquito and these factors, making transmission impossible (Rogers and Randolph, 2006).

Seasonality in Nigeria is characterized with a period of rainfall (March - October) and a period of dryness (November – February). Rainfall is the major key factor to enhance the malaria transmission in several countries (Thomson et al., 2005). Anopheline mosquitoes breed in water habitats, thus requiring a specific amount of precipitation in order to successfully reproduce. Too much rainfall, or rainfall accompanied by storm conditions can flush away breeding larvae. Rainfall also affects malaria transmission by its related increase in relative humidity and modification of temperature, which affects where and in what quantities mosquito breeding can occur. Though we had the limitation of measuring rainfall in the study area but it did not deter the study as we broadly categorized the season into three wider seasons to accommodate all seasons. The species among the complexes of Anopheles were not established and this could have been the novelty if we were able to know the exact species among the complexes responsible for malaria transmission in the study area.

In conclusion, current control strategy using Insecticide treated bed-nets largely involve sampling for adult mosquitoes and knowing their susceptibility status to the insecticide used. This control strategy is to contribute to the decline in densities as well as the parasite levels in both the mosquito vectors and the human hosts. Despite the efforts in some other regions, these vectors have continued to escape control because thorough knowledge of their behaviour, population dynamics, species that are the primary transmitters in a particular area and how they interrelate with their environment have not been studied and documented. A general understanding of seasonal variation, behaviour of Anopheles mosquito and the effect of micro - climatic factors in the abundance of vectors as seen in this study will form the pivot of malaria vectors control and consequently malaria. The findings in the study therefore gives a direction to take in the control of malaria vectors in Ahani Achi, Oji River of Enugu Sate, knowing the seasonal variation of the malaria vectors, their biting behaviour, climatic exposure and the likely human behaviour that encourages the biting tendency of the mosquito.

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