Species Composition, Abundance and Population Structure of Malaria Vectors in Two Villages of Sudan


 BackgroundFrequent monitoring of mosquito vector population is a strategy of great importance for reducing risks of disease occurrence. In Sudan, malaria is still a big threat to public health. Insecticide-based control has been undertaken for years, but there is no noticeable decrease of malaria infection nationwide.ObjectiveTo overcome this situation, a better understanding of the breeding ecology of the vectors is relevant. Here, we investigate the species composition of malaria vectors, seasonal abundance and population structure in two different villages.MethodologyMonthly samplings were performed in Abu Algoni (Sennar State) and Algerif West (Khartoum State) from June 2010 to May 2011. During each visit, immature stages were collected from potential breeding sites using dipping technique. In addition, adults were collected indoors from houses by aspiration and indoor pyrethrum spray methods. Mosquitoes were identified morphologically, the Anopheles gambiae complex diagnosed using PCR and the physiological status of females determined based on appropriate techniques. Environmental parameters namely temperature, rainfall and humidity were measured.ResultsA total of 4,932 mosquitoes comprising of 3047 larvae and 1885 adults (males and females combined) were sampled. Of these, 88.9% were collected from Abu Algoni while 11.1% were from Algerif West. Two species, An. rufipes and An. arabiensis were encountered. Anopheles rufipes was only found in Abu Algoni, while the latter was found in both villages, where it represented more than 99% of the total collection.Mosquitoes were found breeding in many types of places including canals, temporary pools of water, animal hooves, water from broken pipes, and water storage containers. No significant correlation was found between female and temperature (p>0.05). Significant correlation difference was observed between number of females of An. arabiensis and rainfall (p<0.05) and humidity (p<0.01).ConclusionsAnopheles arabiensis is the only member of An. gambiae complex detected in the present study. Seasonal abundance of An. arabiensis was observed with most during the wet rainy season in both villages. This could be associated with the availability of more breeding sites created by the rainfall. The majority were parous which indicates high survival rates and thus high vectorial capacity in transmitting malaria.


Abstract
Background Frequent monitoring of mosquito vector population is a strategy of great importance for reducing risks of disease occurrence. In Sudan, malaria is still a big threat to public health. Insecticide-based control has been undertaken for years, but there is no noticeable decrease of malaria infection nationwide.

Objective
To overcome this situation, a better understanding of the breeding ecology of the vectors is relevant. Here, we investigate the species composition of malaria vectors, seasonal abundance and population structure in two different villages.

Methodology
Monthly samplings were performed in Abu Algoni (Sennar State) and Algerif West (Khartoum State) from June 2010 to May 2011. During each visit, immature stages were collected from potential breeding sites using dipping technique. In addition, adults were collected indoors from houses by aspiration and indoor pyrethrum spray methods.
Mosquitoes were identi ed morphologically, the Anopheles gambiae complex diagnosed using PCR and the physiological status of females determined based on appropriate techniques. Environmental parameters namely temperature, rainfall and humidity were measured.

Results
A total of 4,932 mosquitoes comprising of 3047 larvae and 1885 adults (males and females combined) were sampled. Of these, 88.9% were collected from Abu Algoni while 11.1% were from Algerif West. Two species, An. ru pes and An. arabiensis were encountered. Anopheles ru pes was only found in Abu Algoni, while the latter was found in both villages, where it represented more than 99% of the total collection.Mosquitoes were found breeding in many types of places including canals, temporary pools of water, animal hooves, water from broken pipes, and water storage containers. No signi cant correlation was found between female and temperature (p>0.05). Signi cant correlation difference was observed between number of females of An. arabiensis and rainfall (p<0.05) and humidity (p<0.01).

Conclusions
Anopheles arabiensis is the only member of An. gambiae complex detected in the present study. Seasonal abundance of An. arabiensis was observed with most during the wet rainy season in both villages. This could be associated with the availability of more breeding sites created by the rainfall. The majority were parous which indicates high survival rates and thus high vectorial capacity in transmitting malaria.

Background
Malaria is a major health problem in Sudan leading to morbidity and mortality. Symptomatic malaria accounts for 7.8% of out-patient clinic visits and approximately 12.2% of hospital admissions [1]. About 87.6% of malaria cases in Sudan are due to Plasmodium falciparum, while P. vivax accounts for 8.1%. In North Darfur, West Darfur, South Darfur, River Nile and Khartoum states P. vivax and mixed infection of P. falciparm and P. vivax can reach more than 15%. Transmission occurs all year round in the south but is more seasonal in the Northern State, peaking at the end of the rainy season [1].
Thirty one species of Anopheles have been identi ed but only a handful are malaria vectors [2]. Anopheles gambiae and An. funestus, are important malaria vectors in the southern parts of Sudan and their vectorial capacity may parallel that of An. arabiensis [3] which is considered to be the main malaria vector in Sudan [4]. Its distribution extends from the south up to the borders with Egypt [5][6]. Other anopheline mosquitoes present in Sudan such as An. nili and An. ru pes are of no medical signi cance due to their predominant zoophilic tendencies and their extremely low densities even during rainy season [2]. In the poor savanna area of Central Sudan, it has been found that Anopheles mosquitoes disappear during the dry months of the year and reappear during or soon after the rst rainfall [7].
Anopheles arabiensis shows a remarkable tolerance to water shortage and low humidity [8][9]. This vector was documented to even breed during the dry season in areas along the western bank of the White Nile between Khartoum and Jabel Aulia [7]. During the periods of aestivation, often lasting several months, An. arabiensis may take several blood meals, but the gonotrophic maturation is arrested or proceeds very slowly. The density of this vector reaches maximum during the rainy season especially in irrigated areas [3]. Anopheles arabiensis occurs in desert areas, but is also associated with rivers in Niger, Mali and the River Nile in Sudan [10].
In Sudan the use of insecticides is the most important strategy for controlling malaria vectors through indoor residual house spraying (IRS) and more recently, the use of insecticide-treated bed nets (ITNs) [11]. Current surveys of An. arabiensis show high levels of DDT, malathion, Fenitrothion, Bendiocarb, Propoxur, deltamethrin, lambdacyhalothrin and permethrin resistance in the eastern Sudan, El Rahad, Gezira and Central Sudan [12][13][14][15].
This study aims to gather information on the species composition and seasonal abundance of vector Anopheles in Algerif West and Abu Algoni in Sudan. Adult females collected were dissected for parity. A survey of the larval population was also carried out.

Material And Methods
Two study areas selected for study were: Algerif West: This study was performed in the Algerif West Farm lies on western bank of the Blue River Nile in Khartoum state (latitude 15º 35`394 N and longitude 32º 35' 160 E). This farm has citrus fruit trees, cattle and chicken farming. It is irrigated from water supply from the Blue River Nile. The land is composed of fertile at clay soil.

Abu Algoni village:
Abu Algoni is a village in Sennar State (latitude 13º 31N and longitude33 º E. The soil, mainly alluvial, is naturally very fertile. The main economic activity is agriculture of sorghum and cotton through the irrigated scheme of Suki, the sugar factory of Sennar and a number of fruit farms (including bananas and mangos) located on the banks of the Blue Nile.

Entomological survey
Detection of Anopheles breeding sites: Detection of mosquito breeding sites in and around Algerif West and Abu Algoni villages took place during the study period. All possible sites were visited including the surrounding farms, and the presence of larval anopheline mosquitoes were checked by dipping or netting collection methods.
Mosquito sampling techniques: Anopheline populations at Algerif West and Abu Algoni villages were sampled over 12 months beginning in June 2010 to May 2011. Larval surveys and spray captures of resting adults inside houses were done once a month throughout the period following the standard procedures [16]. Three mosquito collectors assisted in the eld work throughout the study.
Larval collection: Dipping collection method Larval samples of various life stages were routinely collected every month from a xed productive site near the main waterworks of the village from water pools created by the running water, water draining into the vegetation from canals as well as from animal hooves.
Standard dipping collection using a ladle of 8 cm diameter and 3 cm deep with a metal handle of 50 cm length was used. Ten random dips were taken from the xed breeding site for every sample. The ladle was lowered gently at an angle of about 45º until one side was just below the surface. Then the samples were collected in large plastic containers using a pipette and aquatic natural predators (dragon ies, water beetles, tadpoles, etc.), when found were removed. The water samples were then transferred to the laboratory.

Adult collection
Two methods were used for the collection of adult Anopheles mosquitoes resting inside houses. These were indoor spray collection and aspiration collection methods.
Indoor spray collection method: Indoor spray collection (pyrethrum spray collection or knock down collection) method was carried out early in the morning, usually between 6-10am. For this collection 10 houses were randomly selected as xed capture stations, taking into consideration the following criteria; each house is occupied by a number of people and is near to one or more of mosquito breeding sites. People were rst requested to leave the house and then the whole oor surfaces as well as beds and other areas were completely covered from wall to wall with white cloth sheets. All windows, door, eves and other openings through which mosquitoes could otherwise escape were rmly closed. The house was lled with mist of 0.2% solution of pyrethroid diluted in kerosene using hand atomizer (spray pump) at minimum capacity. After spraying, the house was kept closed for 10 minutes and then opened to collect mosquitoes found on the sheets.
Dead mosquitoes were placed into a plastic cup lined with moist cotton wool and covered with a damp lter paper. Samples were transported to the laboratory for examination.

Aspiration method
Resting adult females of morphologically identi ed An. arabiensis from unsprayed houses were aspirated using sucking tubes (aspirator). The aspirator consisted of a glass tube 24 cm in length and one cm internal diameter. A ne mosquito net was xed on the one end of the tube. This end of the tube was inserted into a piece of rubber 60 cm long. The mosquitoes collected were emptied in a plastic cup covered with mosquito net with a central hole plugged with a wet piece of cotton wool. Samples were transported to the insectary, well protected to prevent mortality.
Laboratory techniques: Adults and larval mosquitoes were morphologically identi ed to species level using the morphological keys of Gillies and De-Meillon [17] The numbers were counted for An. arabiensis.
Specimens were classi ed based on the abdominal appearance according to the Sella scale. The ovaries were dissected following the procedure described by WHO [16] to determine the parity rate.
PCR assay for species identi cation PCR analysis was conducted for species identi cation using the rDNA-PCR method because individual species within the Anopheles gambiae species complex cannot be precisely identi ed by morphology alone [18].
Data collection and analysis All An. arabiensis females were eye-checked for physiological status and the resulting numbers of fed, unfed, halfgravid and gravid counted, taking into account location and season of collection. These numbers were used to calculate overall and seasonal mean feeding rates for both villages as follows: (number of fed females + half-gravid / the sum of the numbers of fed + unfed + gravid females) x 100.
Fed and unfed females were also dissected under a binocular microscope for their ovaries and their parity status, determined based on the Detinova method as described by Gillies [19], which relies on the presence or absence of coiled tracheolar skeins in fresh ovaries. The parity rate was calculated as the (number of parous females /number of parous + nulliparous females) x 100, scored population age. All data collected during the study was analyzed using the computer program SPSS (Statistical Package for Social Science) for windows version 18.

Results
PCR results in gure1 showed that all anopheles were identi ed as An. arabiensis. Table 1 shows the Anopheles collections and their seasonal variations in Abu Algoni and Algerif West. A total of 4,587 mosquitoes were collected during this survey, 73.5% of which originating from Abu Algoni. In this latter village, 59.4 (2,007/3,377) and 40.6% (1,370/3,377) of the mosquitoes collected were at the larval and adult stages, respectively. In Algerif West, the larval population collected (85.9% = 1,040/1,210) was much higher than that of adults (14.1% = 170/1,210). The population sizes of the two developmental stages exhibited different seasonal variation patterns according to village. In both villages, the larval population size increased sharply from cool dry season (CDS), reaching peaks during rainy season (RS); however, there was far more larvae in Abu Algoni during hot dry season. (HDS). In both villages, more than 80% of the total adult collections resulted from pyrethrum spray method. In Abu Algoni, the adult population size gradually increased when progressing from HDS to CDS to RS, which recorded 75.8% (1,039/1,370) of the collections. Similar variation pattern of abundance was observed in Algerif West, but there were far more adult mosquitoes in Abu Algoni.  It is interesting to note that in both villages, the number of An. arabiensis gravid females increased as season progressed, but the seasonal numbers of such females were far higher in Abu Algoni. Figure 2 shows that adult abundance was relatively high during the period June-September 2010, and peaked in October in Abu Algoni. Their abundance sharply decreased thereafter and mosquito adults were nearly imperceptible from January to May 2011. In Algerif West as shows in Figure 2, mosquito abundance was very low from June to December 2010 and adults were unnoticeable from January to May 2011.
In Abu Algoni, larval abundance in 2010 gradually increased from June, peaked in August and progressively decreased up to 3 individuals in December of the same year. The larval populations rebounded during early 2011, with major peak (100 individuals) recorded in April (Figure 2). In Algerif West, a similar variation of larval abundance was observed, but the peak was attained in September and was minor compared to that of Abu Algoni. In Algerif West, larval abundance was very low in January 2011 and larvae were not found thereafter (Figure 3).
The parity of An. arabiensis exhibited different variation patterns between Algerif West and Abu Algoni. In the rst village, this parameter increased from June to attain a rst peak in August. A similar trend was observed in Algerif West, but the number of parous An. arabiensis was lower compared to Abu Algoni. In this latter village, the parity rate recorded a second peak in August thereafter until October, a time when the number of parous females peaked in Algerif West. In Algerif West as well as Abu Algoni, the number of parous females increased from November 2010, attaining major peaks in January 2011. The parity rate in both villages sharply decreased in the next month. No An. arabiensis was found for the rest of 2011 in Algerif West, whereas parous females were still present in considerable numbers from March to May of the same year ( Figure 4).

Correlations between An. arabiensis abundance (larval and female) with-climatic factors
In Abu Algoni, An. arabiensis female populations varied considerably from one month to another, as did measured climatic factors. From June to July 2010, the number of females increased with increasing rainfall, but decreased in August, the month that recorded the highest rainfall. After August 2010 the female population rebounded and peaked in October. During this period (August-October 2010), there was much less rainfall and the environment was drier. At the end of the rainy period (November), the number of females was low and remained almost contant when rains were scarce and humidity low (December-May 2011) ( Figure 5 ). Larval populations in Abu Algoni (AA), moisture conditions were low and almost constant during the survey period, except the slight increase observed from July to September 2010. Furthermore, it often rained during the early months of the survey (especially in July and August 2010); however, there were no rainy days thereafter. Concomitant with increased rainfall events and relative humidity, mosquitoes were present in large numbers, in particular larvae. Larval populations sharply decreased from August towards September 2010, a period that corresponded with decreasing rainfall events. This, in combination with the reduced mosquito populations (larvae and adults) from November 2010 to May 2011, is likely to suggest rain as a driving force of mosquito breeding in this village ( Figure 5).
In contrast to Abu Algoni, there was not marked variations of climatic factors in Agerif Wesy. Here, An. arabiensis females maintained a low and nearly invariable population size throughout the study (Figure 6). A similar trend of larval population (Figure 6) variations were observed in Algerif West (AW) but with several differences; (i) a lower rainfall throughout 2010, (ii) the peak of larval abundance was recorded later (September), (iii) larvae were less numerous and (iv) absence of larvae when no rain event occurred (2011).

Discussion
Our study revealed that An. arabiensis is the predominant species found and is also the only member of the An. gambiae complex observed at the two study sites. In Abu Algoni, all larvae collected were identi ed as An. arabiensis. Except for several An. ru pes individuals encountered, all trapped adults were also from the same species. Similarly, at Agerif West, only An. arabiensis was found. Overall, the prevalence and abundance of both larval and adult populations exhibited a seasonal pattern and were in uenced by changes in the monitored climatic parameters. Finally, there were increased parity rates, thus indicating aged An. arabiensis populations in both villages.

Species composition
Throughout the survey, we observed increased prevalence of An. arabiensis at both larval and adult stages in AL and AW. This is in line with a report from Osman [20] who surveyed the Anopheles populations in Sennar State. Observed that 92% of collected Anopheles individuals were An. arabiensis and considered it as the main malaria vector in the area. This species also accounted for more than 99% of total anophelines collected in AL, which is also located in Sennar. Similarly, Himeidan et al., (2004) showed that An. arabiensis is the main malaria vector in Kassaala State. Seidahmed et al., [21], Yagoop et al., [15] and [22] con rmed that An. arabiensis is the main malaria vector in Khartoum, El Rahad Central Sudan and in Ed Dueim, White Nile State respectively.

Variations in population abundance relative to climatic factors
Algerif West area is characterized by a low density of An. arabiensis and restricted only to the rainy season. This reduction has been accomplished by the e cient control activity in Khartoum State through the Malaria-Free Khartoum programs.
The population density of An. arabiensis was monthly recorded during the study period except in June. The major peak was recorded in October and two minor ones in February and April 2011. Our current study revealed that there is a direct correlation between the amount of rainfall and density of An. arabiensis. This study shows that the survival and development of large range due to the climatic and environmental factors. The high abundance of An. arabiensis during the rainy season could be associated to the availability of more breeding sites created by the rainfall. Interestingly, there was a reduction in female mosquitoes during August although this is the wettest month. This observation could be attributed to the cleansing of the breeding sites which effectively washes down the larvae and the eggs, consequently reducing the abundance of adult An. Arabiensis. This is in agree with the ndings of Adeleke et al, [23].

Population structure
Mosquito age composition plays an important role in malaria transmission. Parous females that have previously blood fed, have a higher possibility of being infected with malaria parasites. Lemasson et al. [24] conducted a study comparing the behavior and vector competence of An. gambiae and An. arabiensis in Senegal during 1994-1995. They observed that An. gambiae had a higher parity rate than An. arabiensis, but data collected during [1995][1996] showed that the parity rate of An. gambiae was signi cantly lower than An. arabiensis [24]. These ndings are similar to the current study where the parity rate was high in An. arabiensis in the two study sites in Sudan. Ndiath et al. [25] studied the dynamics of transmission of Plasmodium falciparum by An. arabiensis and the molecular forms M and S of An. gambiae in Dielmo, Senegal. They found that the mean parity rate was 70.9% for An. arabiensis, 68.7% and 80.1% for An. gambiae M and S forms, respectively. These ndings are in line with the present study where the mean parity rate was 77.19% in Abu Algoni village and 63.85% in Algerif West village. The low parity rate in Algerif West village maybe due to the effective vector control programme in this area. In contrast Himeidan et al. [13] who studied the biology and behaviour of An. arabiensis in New Halfa eastern Sudan recorded a parous rate of 32.23%. He explained that the low level of parity rate might be due to the application of insecticide during the study period.

Conclusions
In both villages, the larval population size increased sharply from CDS, reaching peaks during RS; however, there was far more larvae in Abu Algoni during HDS. In Abu Algoni, the adult population size gradually increased when progressing from HDS to CDS to RS. Similar variation pattern of abundance was observed in Algerif West, but there were far more adult mosquitoes in Abu Algoni. This study has established that An. arabiensis is the most important vector of malaria in Sudan. Anopheles populations are naturally subject to environmental factors in which they inhabit. These factors play a major role in controlling their population dynamics, population genetic structure and their effectiveness as vectors. The climatic changes in Sudan would predictably cause uctuations in the An. arabiensis populations. This would ultimately affect the population dynamics and population genetics of these vectors. Mosquito age composition plays an essential position in malaria transmission. Parous females that have previously blood fed; have a higher opportunity of being infected with malaria parasites and then transmitting the disease. Availability of data and materials The data supporting the results reported in this article are included within the article.

Competing interests
The authors declare that they have no competing interests