1. Introduction

It is estimated that globally, 1.2 billion people are at risk to lymphatic filariasis (LF), major cause of acute and chronic morbidity affecting humans in tropical and subtropical countries[1,2]. The absence of a nonhuman reservoir, availability of safe, single-dose, two-drug treatment regimes, capable of reducing microfilariae to very low levels and remarkable improvement in techniques for diagnosing the disease, resulted in advocacy for a global strategy to eliminate filariasis through mass drug administration (MDA)[3]. This culminated in a global alliance by the World Health Organization and other agencies to eliminate lymphatic filariasis by 2020[4]. The main goal is to break the cycle of transmission of the parasites, between mosquitoes and humans mainly through MDA with Albendazole in combination with either Ivermectin or Diethylcarbamazine citrate[5]. Although there had been progress since the initiation ofMDA Programmes, challenges have emerged. These include: the inability of some countries to sustain MDA, non- availability of data to indicate level and duration of treatments to eliminate LF, and a shift towards linking MDA for LF control and other neglected diseases[6]. These challenges have led to growing concerns on the effectiveness of MDA alone to eliminate LF, without vector control as a complement[7]. Thus an integrated strategy involving vector control is now thought to have great potential to become an important supplementary component of the filariasis elimination campaign.

Culex quinquefasciatus (Say) is an urban vector of nocturnally periodic Wuchereria bancrofti in West Africa[8]. The distribution of the urban vector of bancroftian filariasis, Cx. quinquefasciatus is expanding with urbanization; many rural areas that were free of this vector are now being colonized[9]. Reliable data on breeding sites of species ensure the effectiveness of new control methods. Nicolas et al.[10] controlled this species, by applying Bacillus sphaericus against the immatures at breeding sites; they estimated that this approach would be more cost effective than the use of chemical insecticides. There had been reports of cases of bancroftian filariasis in rural Local Government Areas (LGAs) adjacent to the Ikwerre LGA of Rivers State, Nigeria[11,12]. Records indicate that patients had been treated for filariasis in the General hospital at Isiokpo, Ikwerre LGA headquarters. Studies were therefore undertaken, June- September, 2009 (Rainy season) in 5 villages (Ozuaha, Ipo, Omanwa, Omademe, Ubima) in Ikwerre LGA, Rivers State, Nigeria. These villages were selected by the State Government for a World Bank-assisted project on Integrated Vector Management (IVM) for malaria control. Studies on Cx. quinquefaciatus were undertaken simultaneously with those on Anopheles spp. to determine whether Cx. quinquefasciatus described as primarily urban (service 2008) had invaded rural areas and to identify their breeding sites.

2. Materials and Methods

2.1. Study Area

The 5 villages IPO (06^º57.5´N, 05º02.3´E), OMANWA (06º53.5´N, 05º03.8´E), OZUAHA (06º55.3´N, 05º03.5´E), OMADEME (06º57.5´N, 05º05.1´E) and UBIMA (06º54.2´N, 05º07.4´E) are located in the lowland rainforest. Ubima and Ozuaha were more developed (paved roads and modern housing) while Ipo and Omanwa were the most rural. There are 2 seasons April-September (rainy) and October-March (dry). The main occupation of the local people is farming; some are involved in wildlife hunting and bush meat trade.

2.2. Methods

A 100 x 100m grid was randomly selected in each village. Potential breeding sites were sampled with a 100ml-laddle and a Pasteur pipette for sites with large and small volumes of water respectively, daily for 4 months, June-September, 2009. These sites included Phytotelmata (water-containing holes in plants), Pools (puddles, gutters) and containers (water receptacles in villages, such as the calabash, from the fruit of Cresenta cujeta Linn, Bignonaceae), plastic and metallic containers. Culex larvae were identified by the descriptions in Service[13]. Immatures were reared to adults to confirm that they were those of Cx. quinquefasciatus as per the keys of Gilles and de Meillon[14] and Gillett[15]. Anopheles gambiae s.l. immatures were also collected and reared to adults during the study. Adults of An.gambiae s.l. were identified by the keys of Holstein[16], Gillett[15] and Louis[17]. Numbers of immatures were recorded as per breeding site-types and villages. Analysis of Variance with one observation per cell was used for statistical analyses. Calculated F values were compared to tabulated F values to determine significance.

3. Results

3.1. Diversity and Yield of Breeding Site-Types

The total number of Cx. quinquefasciatus immatures was approximately 6-fold that of An. gambiae s.l. immatures. Nearly 80% of all immatures were collected from container-type breeding sites (metal and plastic containers, “calabash”, and tyres) and least (about 5%) from phytotelmata (Table1). The differences were significant (F_Cal=35695636; F_Tab=39.86; df 1,1; p<0.01). The highest number of immatures per breeding site was also in the container category (Table 2); the differences were significant (F_Cal=16.5; df 1,3; p<0.05). Approximately 80% were from plastic containers and least 1% from tyres in the container-type breeding sites. The differences were significant (F_Cal=4.7 x 10.8; F_Tab=8.53; df 1,2; p<0.01). The highest number of immatures per breeding site was from calabash in the container-type (Table 3); the differences were significant (F_Cal =23.1; df1, 2; p<0.05). In the pools breeding site- type, approximately 55% of immatures were from puddles and the rest from gutters; the difference was not significant (F_Cal=334975; F_Tab=39.86; df 1,1; p>0.01), but the difference in the number per breeding site between puddles and gutters was significant (F=18.16; df 1, 3; p<0.05). Percent occurrence of Cx. quinquefasciatus immatures varied among breeding site categories: containers (69.23-100.00%), Pools (66.67-76.47%) and phytotelmata (75.0-82.93%) (Table4). In the phytotelmata breeding site-type, about 67% of immatures were collected from depressions on trees; the difference in total numbers among site-types was significant (F_Cal=2.47 x 10⁸; F_Tab=8.53; df 1,1; p<0.01).

3.2. Variation in Site-Type Utilization across Villages

At Ipo and Omanwa, about 90% of immatures were from container-type breeding sites; this figure decreased to 60% at Omademe. In contrast, at Ozuaha, nearly 70% of immatures were from pools, while at Ubima more than 80% were from phytotelmata. The variations in breeding site preference patterns among villages were significant (F_Cal =4305.94; F_Tab=39.86; df 1,1; p<0.01).

Table I. Numbers of Immature Culex quinquefasciatus from various breeding site-types

Table 2. Numbers of Culex quinquefasciatus Immatures at Different Breeding Sites in the Five Villages

Table 3. Numbers of Culex quinquefasciatus per Breeding Site Site-Type Categories Breeding Site- types Number of Breeding Site Examined Number of Immatures Collected Number per Breeding Site Containers Tyres 9 16 1.78 Plastic 43 3570 83.02 Metal 12 584 48.67 Calabash 2 486 243.00 Sub-total 66 4656 70.55 Pools Gutter 24 408 17.00 Puddle 13 487 37.46 Sub-total 37 895 24.19 Phytotelmata Depression on tree 6 196 32.67 Plantain/ Banana Axils 34 97 2.85 Sub-total 41 293 7.33 Total 144 5844

Table 4. Percent Occurrence of Culex quinquefasciatus at Breeding Site-types

4. Discussion

The high numbers of total Cx. quinquefasciatus larvae collected in the villages, indicated that the species, once considered an urban species[18, 13] is increasing in distribution and colonizing rural pockets that were once free of this mosquito, an aspect of Cx. quinquefasciatus distribution that was also observed by Chavasse et al.[9]. In urban areas, the typical breeding sites were described as stagnant polluted water and in rural areas, mainly privies (pit latrines)[18, 13]. The extensive distribution of breeding sites (tyres, plastic and metal containers, calabashes, gutters, puddles, leaf axils, depressions on trees, etc) is an indication of changes in the oviposition behaviour of Cx. quinquefasciatus; pit latrines, typical of polluted habitats did not occur in the randomly selected grids. The dominance of containers as breeding sites of Cx. quinquefasciatus had been reported by Hardine et al.[19] and Bockarie et al.[8].

Potential capacity to enhance offspring survivorship and fecundity contributes by definition, to parental care or juvenile dispersal; offspring survival and growth may depend strongly on the quality of the habitat in which they are deposited. Thus, when potential habitats vary in their suitability for juveniles, females are expected to choose habitats that maximize fitness[20]. In mosquitoes, such oviposition habitat selection was demonstrated in response to physical and chemical suitability for larval development[21]. This may account for the variation in preference among metallic, plastic containers and, calabashes. The low numbers of immatures in phytotelmata site-types may be associated with habitat size and limited resource availability[22-24] .

Although container-type sites were preferred at Omanwa, Ipo and Omademe, pools and phytotelmata were the selected sites at Ozuaha and Ubima respectively. The pronounced preference for containers at Ipo and Omanwa might be attributed to the extreme rural conditions in the villages, limited access to running water and therefore utilization of containers for collecting and storing rainwater. Unfortunately, these containers served as mosquito breeding sites. The dominance of phytotelmata at Ubima might be attributed to the extensive occurrence of plantain/banana groves in the randomly selected grids. These observations indicate that variations are common and therefore breeding site preference studies should be undertaken in each locality before the commencement of larviciding. The ubiquitous nature of Cx. quinquefasciatus breeding sites is highlighted by its high percent occurrence in all site-type categories.

5. Conclusions

Cx. quinquefasciatus, long considered an urban species has invaded the rural lowland rainforest. Most immatures were collected from container-type breeding sites (metal and plastic containers, “calabash”, and tyres) and least from phytotelmata. The highest numbers of immatures were collected from plastic containers and calabashes. Degree of breeding-site utilization varied across villages.

ACKNOWLEDGEMENTS

This study was undertaken during a World Bank-funded project on the Indoor Residual Spray (IRS) - Integrated vector Management (IVM) for malaria control. The beneficiary was the Rivers State Ministry of Health, Port Harcourt, Nigeria. We are indebted to village chiefs, who granted us access to the study sites and the youths that served as guides in each of the villages. The commitment of Messrs Pasiya Otufu, Stephen Ozule and Ibrahim Sow Kamara during field work is appreciated. The assistance on statistical analyses by Mr Bukola Amoo Oyebade of the Forestry Biometrics and Measurement Unit, Department of Forestry and Wildlife Management, Faculty of Agriculture, University of Port Harcourt is gratefully acknowledged.