Seasonal abundance and molecular identification of West Nile virus vectors, Culex pipens and Culex quinquefasciatus (diptera: culicidae) in Abeokuta, South-West, Nigeria.

BACKGROUND
West Nile virus (WNV) infection, is an arbovirus infection with high morbidity and mortality, the vector responsible for both human and animal transmission is Culex pipens complex.


OBJECTIVE
To determine the species distribution and seasonal abundance of Culex pipens and Culex quinquefasciatus mosquitoes in Abeokuta, Nigeria.


METHODS
Mosquitoes belonging to the Culex pipens complex were captured in three different locations located within Abeokuta Metropolis between March 2012 and January 2013. Individual species were identified using morphometric methods. Amplification of the Ace2 gene by PCR confirmed morphormetric identification of the mosquitoes.


RESULTS
A total of 751 mosquitoes were captured. Culex quinquefaciatus recorded the highest distribution of vectors with 56.6% and Culex pipens 43.4% (P > 0.05). Idi aba community recorded the highest distribution of mosquito vectors with 42.9% (n=322) and Culex quinqueaciatus was more abundantly distributed with 183 mosquitoes. Aro community recorded 32% (n=240) of captured mosquitoes with Culex quinquefaciatus having a higher level of abundance and lastly Kemta with a distribution of 25.1% (n=189).


CONCLUSION
Results from this study show that potential vectors of WNV abound within Abeokuta, putting residents at high risk of West Nile infection. We advocate for introduction of routine testing of WNV in Abeokuta and Nigeria.


Introduction
Mosquitoes (Diptrea) are principal vehicles in the transmission of various diseases, mainly viral and parasitic.Major diseases transmitted are known as Arboviral diseases because these viral infections are transmitted by arthropod vectors 1 .Human and animal pathogenic arboviruses include, WestNile virus, (WNV), Yellow fever virus (YFV), Dengue virus (DNV) and Rift valley fever virus (RVFV) 1 .In Africa where climatic conditions and vegetation promotes abundant reproduction and distribution of diverse mosquito species, large epidemics of Arboviruses have occurred for instance the Yellow ever epidemic in Nigeria during the mid-1980 which ravaged thousands of lives 2 .West Nile virus is a highly virulent potentially lethal neurotropic virus widely distributed in the Americas and Europe 3,4 .In Nigeria, WNV infections have been reported 5,6 .The maintenance and spread of WNV is dependent on the relative abundance of the insect vector Culex pipens 7 , Humans are accidental or secondary host of WNV, birds and horses are the primary hosts of this virus and maintain the virus in nature.For outbreaks to occur the primary hosts must be viremic 1 .Vectorial studies have demonstrated the presence of West Nile virus in Culex pipens complex mosquitoes in Nigeria 6 .Apart from WNV, Culex quinquefasciatus is one of the major vectors of Wuchecheria brancrofti infection world-wide, a parasitic infection having various disease manifestations 8 .
A recent study reported Brancroftain filariasis infection among inhabitants of Abule Sowo, a remote village few kilomerters from Abeokuta 9 , Nigeria.Previous studies have also demonstrated the public health implications of high vectorial abundance of Culex pipens complex in Abeokuta 10 .However only Morphometric analysis has been done in the identification of Culex pipens complex 11,12 , the molecular identification of this all important insect vector has not been attempted in our environment, despite its use in surveillance and experiment studies elsewhere.There have been reports on the limitation of only morphometric methods in the identification of various Culex species in Nigeria 8 .With reports of existence of hybrid spp of culex pipens complex and misidentification of mosqutoes within the culicine family particularly Culex 13,7 .There is need to introduce Molecular biology methods to validate existing morphological classification keys as well as identify hybrid spp in mapping out the distribution and abundance of Culex spp in Nigeria.The aim of this study was to use Molecular method (PCR) to identify and diferentiate Culex pipens complex and determine the vectorial abundance and seasonal distribution of the vector in some selected residential areas within Abeokuta Metropolis.

Study area
The current study was conducted at Abeokuta, Ogun State, Nigeria.Abeokuta is the capital city of Ogun State, with co-ordinates, latitude 7010'N and longitude 3021'E, located in the transitional zone between tropical rain forest and derived savannah in South-Western Nigeria.The city is a rapidly developing city with increasing population density and increased construction of civil infrastructures within the city.Abeokuta is inhabited by the Yoruba speaking tribe in South-Western, Nigeria.There are 2 major seasons, the dry season (November to March) and the rainy season (April to October).The study locations were in 3 urban residential areas located within 2 Major local government areas in Abeokuta, Federal Medical Centre Idi-aba, Kemta estate Located in Abeokuta South Local government area, and Aro community, located in Abeokuta North local government area.

Mosquito capture and identification
Adult mosquitoes were captured using baiting method in sterile sample bottles by trained volunteers, in 4 randomly selected spots around each selected study location.Volunteers for sample collection presented evidence of vaccination against yellow fever, and were put on Malaria prophylactic treatment throughout the duration of mosquito field collection.Mosquito capture was done between the hours of 3 pm and 8 pm.Upon capture Culex mosqui-toes were sorted out by species from other insects using morphorlogical keys as described by Gillet 14 .Male Culex mosquitoes were morphologically identified by their male genitalia under the microscope 15 .Female Culex pipens and Culex quinquefasciatus mosquitoes were morphologically differentiated by using wing measurements' of intersection of costa, subcosta and bifurcation of R2+3 veins, as described by Antunes de Morias et al 16 , Dehghann et al 17

DNA extraction
Individual Culex mosquitoes previously identified into individual species by morphometric methods were selected according to location of capture, and ground in a sterile porcelain mortar and pestle in serum free, minimum essential medium (MEM).Genomic DNA was extracted from the homogenate using DNA extraction kit from Jena Bioscience® Gmb Germany.

PCR
Molecular identification of Culex pipens, and Culex quinquefascius was done by multiplex polymerase chain reaction (PCR), amplifying the Acetylcholinestarase gene locus (Ace2 gene) of the mosquito, using a set of previously published primers 18 , in an Applied biosystems (ABI 9700) genetic analyzer Foster city CA.The primer set are ACE quin, 5'-CCTTCTTGAATGGCTGTGGCA-3', ACEpip 5'-GGAAACAACGACGTATGTACT-3', B1246s 5'-TGGAGCCTCCTCTTCACGGC-3'.The cycling condition was one cycle for 950C for 5 mins, followed by 35 cycles of 950C for 30 secs, 550C for 30 secs 720C for 1 min, and a final extension step of 720C for 5 mins.The expected band size is 610-bp for culex pipens and 274-bp for culex quinquefasciatus, and both bands showing in hybrid mosquito species.

Data analysis
Results from field and laboratory studies were recorded, grouped and analyzed using statistical software SPSS 12.0.

Results
A total of 751 mosquitoes belonging to the genera culex were captured.Distribution of mosquito species showed that Culex quinquefasciatus was the most abundant with 56.6% (n = 425) and Culex pipens with 43.4% with P > 0.05.Majority of the mosquitoes were female.The seasonal abundance of mosquito vector species peaked between the months of June and July, with Culex quinquefasciatus having a higher distribution of vectors (Figure 1).
African Health Sciences Vol 16 Issue 1, March 2016 However there was a decline in vector abundance in the month of August which rose again in September.There was a gradual decline in the abundance of both species through the dry months of November to January. Figure 2 shows the gel picture of PCR product of Ace2 gene of Culex quinquefasciatus.Species distribution of Culex pipens complex vectors in the 3 locations namely, Idi-aba, Kemta, and Aro as shown in figure 3. Idi aba recorded the highest distribution of both vectors 42.9% (n=322) Culex pipens (139), Culex quinquefasciatus (183).This is followed by vectors caught around Aro community 32% (n=240) with Culex quinquefasciatus having a higher level of abundance.Lastly Kemta with a distribution of 25.1% (n=189).In all the locations Culex quinquefaciatus had higher level of abundance as in figure 3.

Discussion
The current study surveyed the abundance and species distribution of potential West Nile virus vectors, Culex pipens and Culex quinquefasciatus in 3 residential locations within Abeokuta Metropolis.West Nile virus has not been reported in Abeokuta, this is probably due to unavailability of diagnostic facilities capable to detect the infection in Abeokuta, coupled with poor surveillance activity.Previous reports have however indicated the presence of this virus in nearby Ibadan city 6 .This is an indication that WNV is most likely circulating within the whole of the South-West region of Nigeria.Similarly unconfirmed cases of probable West Nile infection have been reported in children at the Federal medical centre Abeokuta (Nwadike, personal communication).
In a recent study of indoor sampled mosquitoes, the WNV vector culex quinquefasciatus was the 2 nd most abundant mosquito sampled in Abeokuta with 32.2% distribution 10 .Our study not only identified Culex pipens complex but also differentiated the 2 main species using both morphometric characters and molecular methods.Culex pipens and Culex quinquefasciatus mosquitoes are almost morphologically indistinguishable, particularly the females making it very difficult for researchers to identify.This was why we adopted the molecular technique, which has been widely used and documented to be highly specific for the identification of these species of mosquitoes and their hybrids 13 .This method employed PCR amplify-ing a fragment of the Acetycholinestase gene locus of the mosquito (Ace2) and sizing the amplicons in agarose gel.This method has also been used to demonstrate the presence of hybrid species between Culex pipens and Culex quinquefasciatus which has served as valuable information in the surveillance and elimination of this vector in the Americas 13,16 .Seasonal abundance of WNV vectors indicated that both mosquito species were more abundant in the months of May to October, coinciding with the rainy season where rainfall and humidity is high.There was however a decline in the relative abundance of both vectors particularly Culex quinquefasciatus in the months of July and August.A similar trend in seasonal abundance has been reported from Abeokuta 10 , where higher level of vectorial abundance was observed in the rainy months of May to October than the dry months, although in the report of Adeleke et al 10 there was a sharp decline in the months of April to July which heralds the peak of the rainy season among Mansonia Africana mosquitoes, the principal vector of plasmodium spp.
A similar trend was observed for Culex quinquefascitus mosquitoes in our current study with a decline in the midrainy season between the months of June and August.It has been suggested that this observation might be due to the heavy rainfall occurring in the peak of the rainy season, thus washing away breeding sites, strand larvae and damage mosquito eggs thereby causing a reduction in the abundance of adult mosquitoes 19 .Species distribu-African Health Sciences Vol 16 Issue 1, March 2016 tion according to location of capture showed that Culex quinquefasciatus was the most abundant species in all three locations sampled.This is in accordance with previous reports which have reported a high level of abundance of Culex quinquefasciatus in relation to other Culex species 10 .No hybrid species were encountered in the current study, although this may not represent the true picture as a major limitation to this study was our inability to consistently test each sampled mosquito using PCR as a result of cost.Only a small proportion of identified mosquito species were tested using PCR.Idi-aba community recorded the highest proportion of mosquitos captured followed by Aro community then Kemta.This observation might be due to the fact that the locations of capture at Kemta were in largely developed residential areas, devoid of potential breeding sites for mosquitoes, unlike areas sampled at Idi-aba where there are various uncompleted buildings and items such as old tyres and cans that can retain water and promote breeding of mosquitoes.The high population of these mosquitoes in areas inhabited by humans gives concern, because of the risk of infection with WNV should an outbreak occur or the possibility of repeated outbreaks where there is presence of vireamic birds or other primary hosts.In the USA, where there is established surveillance for WNV disease, presence of hybrid species between Culex pipens complex, has been associated with rapid spread of WNV 20 .

Conclusion
The current report highlights an overwhelming evidence of the presence of WNV vectors in Abeokuta, Southwest, Nigeria.This reveals that inhabitants of Abeokuta city are at great risk of WNV infection, we advocate for intensified surveillance for WNV, distribution of insecticide treated bed nets, as well as other vector control measures such as clearing of gutters, drains and disposal of unused containers and bad tyres in order to reduce potential breeding sites of mosquitoes within residential areas in Abeokuta and Nigeria.

Aknowledgements
The authors will like to acknowledge the contribution of Dr MA Adeleke for supporting this research with useful materials.The effort of Mr Opayele is also appreciated during the process of mosquito identification.The authors declare that there are no conflicts of interest.

Fig 1 :
Fig 1: Seasonal abundance and monthly distribution of culex pipens and Culex quinquefasciatus mosquitoes in some residential areas in Abeokuta, Nigeria.

Figure 3 :
Figure 3: Bar graphs showing distribution of culex pipens complex in 3 different areas in Abeokuta, Nigeria.