The present survey was carried out to study the typology of breeding sites as well as the physicochemical and microbiological variables favourable to Anopheles larvae development in four cities in Benin. The breeding sites of mosquitoes in different areas were mainly formed as a result of human activities and urbanization related factors. These are excellent breeding habitats for Anopheles larvae. During this study, seventeen different types of habitats have been identified, ranging from puddles to abandoned tyres, including construction sites and vegetable farms. This diversity of habitats for Anopheles larvae has already been observed by Djegbe et al. in 2019 [29] in southern Benin and by many other authors in Africa [30–33]. Puddles were the main breeding sites recorded in the different study sites. These puddles were generally found in alleys or on the outskirts of the streets, which essentially denotes a lack of rainwater sanitation project from the public authorities and a poorly controlled urbanization process in these cities [34, 35]. In addition, the neglect of the local population and the economic activity practices could be the source of many types of habitats [32, 36]. Thus, the abundance of tyres serving as larval habitats for Anopheles in Parakou could be due to the increase in the transport of large trucks which serve other cities in the north and the neighbouring countries, transiting through Parakou. This leads to abandoned tyres in the city's many garages and vulcanization centres.
In addition, the classification of breeding sites as "Natural" or "Artificial" sites enables to highlight the role of humans through routine activities in maintaining the transmission of malaria in the dry season. Indeed, though the number of larval habitats is considerably reduced during this season, those found are mainly anthropogenic. Therefore, it is important to draw the attention of the population on their role in reducing the transmission of malaria through sanitation of their environment and elimination of these shelters [33].
In this study, the larval habitats were also classified as “Permanent” or “Temporary”. This clustering of habitats helps understand their stability and the extent to which they contribute to the abundance of Anopheles populations during seasonal changes in the study areas [37]. A high number of temporary habitat types was observed during the two collection periods in the four cities. As the temporary breeding sites depend mainly on rain and dry out quite quickly due to the drop in precipitation, their high proportions observed in the dry season could be due to anthropogenic activities. These results were in contrast to those obtained by Kudom in 2015 who observed a high proportion of permanent habitats in the cities of Accra and Sekondi-Takoradi during the rainy and dry seasons [36]. On the other hand, these results were in line with those of Koumba et al., (2018) who obtained a high proportion of temporary sites both during the rainy and dry seasons in the agricultural areas of the Mouila zone in Gabon [30].
In three of the four cities surveyed, including Natitingou, Bohicon and Cotonou, the majority of Anopheles breeding sites were found in urban districts. This could be explained by the fact that in urban districts, breeding sites are accessible and easily spotted, unlike districts with low urban potential [29]. In addition, urban districts offer a multiplicity of typical breeding sites (temporary, sunny and clear) favourable to Anopheles, contrary to non-urban districts characterized by unsanitary conditions, and where potential breeding sites found are dominated by pollution less favourable to Anopheles development [38, 39]. These results are interesting because they justify the existence of a fairly high urban transmission of malaria, which a few years ago was still controversial [40].
In Bohicon and Cotonou, tyre tracks were the most productive habitat types. They were the second most productive in Parakou city. This can be explained by the fact that tyre tracks constitute the ideal sites for oviposition of Anopheles females due to their small surface area (0.76 ± 0.36 m2 and 0.7 ± 0.42 m2 in the rainy and dry seasons, respectively), their adequate exposure to sunlight and the absence of plant debris on their surface [41, 42]. Other than tyre tracks, puddles, vegetable farms and gutters were among the habitat types with the highest larval densities in the four cities. Mattah et al., (2017) found similar results with puddles (13.7 larvae/dip), gutters (12.9/dip) and urban farm sites (11.6/dip) as most productive larval habitats of Anopheles in southern Ghana [34].
It is also important to note that the larval abundance of the different types of habitats is mainly influenced by the seasons and the physicochemical parameters. The results showed a seasonal variation in larval densities with higher density in the rainy season compared to the dry season in all types of breeding sites. It is therefore appropriate that the various control strategies to reduce malaria transmission should be intensified during the dry seasons [43].
Moreover, the physicochemical characterization of the habitats made it possible to positively correlate the density of Anopheles larvae with certain parameters including temperature, oxygen level and pH. The positive tropism of Anopheles larvae with temperature has already been shown by several authors [44, 45, 46]. According to Muturi et al. [46], low water temperatures cause a decline in the growth of microorganisms on which mosquito larvae feed. In addition, higher temperatures can be detrimental to the presence of many aquatic arthropods, including predators, increasing the chances of survival of Anopheles larvae. [13]. Temperature could therefore be a factor accelerating the development of larval stages.
Regarding the positive correlation of Anopheles larval density with the dissolved oxygen concentration, similar results were obtained by Mahamane et al. [4] and Mbidda et al. [47]. Anopheles larvae not having a breathing siphon, dissolved oxygen in the water could theoretically be an important factor for their survival. Other authors have shown that anopheline larvae prefer fresh, well-oxygenated water with low mineral content [19]. It should nevertheless be notified an inclination of the Anopheles larvae to salinity in the city of Cotonou. This positive correlation of the density of Anopheles larvae with the salinity observed in Cotonou suggests a tolerance of this species to unusual habitats. Cotonou being a coastal city, its proximity to the sea is believed to be the source of sometimes high salt levels in certain water points in the city, to which Anopheles gambiae would be suitable. In addition, anthropogenic factors, which are even more accentuated in Cotonou due to the high demography and the development of industrial activities which could affect the quality of the water in the larval habitats. These observations are in agreement with the findings of many authors [36, 48], who found that the Anopheles larvae present in brackish roosts in Cape Coast (Ghana) were mainly of the subspecies Anopheles coluzzii. Subsequent work, taking into account molecular identification will be carried out in order to analyse the distribution of the subspecies of the Anopheles gambiae complex within the different types of breeding sites identified.
Microbiological analysis has identified the presence of faecal and total coliforms at various concentrations in the water samples from the breeding sites. The presence of these bacteria in the water indicates faecal contamination of the breeding sites which may be of human or animal origin. Furthermore, the negative correlation was observed between Anopheles larvae and the concentrations of faecal coliforms, suggesting that these coliforms inhibit the growth of Anopheles larvae [19]. While bacteria are known to be a source of nutrients in the growth of mosquito larvae, they may also be indicative of environmental conditions favourable to the presence or absence of larvae in breeding sites [49]. For example, Dada et al. [50] showed that the abundance of Escherichia coli in breeding sites was significantly negatively correlated with the density of Aedes aegypti larvae. Experimental work in the laboratory will be carried out to better understand mechanisms associated with this antilarval activity of faecal coliforms. The results on the potential role of these bacteria in hindering the survival of Anopheles larvae could be useful in different aspects of larval control.