Background

Mosquitoes threaten the live and livelihood of millions of people worldwide (Boussès et al., 2018; Braaks et al., 2018; Carnevale & Robert, 2009; WHO, 2014) because they can transmit diseases such as malaria, dengue, chikungunya, filariasis, encephalitis, Rift Valley fever, yellow fever and Zika fever (Ikram et al., 2016). According to WHO, mosquito-borne diseases account for about 17% of the total burden of all infectious diseases across the world (WHO, 2014). In Cameroon, several studies have been carried out on Anopheles mosquitoes (Antonio-Nkondjio et al., 2005; Antonio-Nkondjio et al., 2019; Bamou et al., 2018; Djamouko-Djonkam et al., 2019; Mbida et al., 2018; Tchuinkam et al., 2011; 2010), the vectors of human malaria, but little is known about the systematics and ecology of other mosquitoes species including those of the genus Culex. Culex mosquitoes are responsible of high nuisance and painful bites (Nchoutpouen et al., 2019) and are known to transmit several diseases such as lymphatic filariasis, Rift Valley fever, West Nile fever and avian malaria (Al-Ashry et al., 2018; Ikram et al., 2016; Ochieng et al., 2013; Schmid et al., 2017; Viniaker & Lavaud, 2005). These mosquitoes are largely distributed in urban settings of Cameroon (Nchoutpouen et al., 2019; Ngadvou et al., 2020) where previous studies indicated circulation of arboviruses in Culex mosquitoes species (Brottes et al., 1996; Salaun et al., 1969).

Like most of the large agglomerations in the intertropical zone, the cities of Dschang and Santchou are undergoing a rapid demographic growth due to population displacement resulting from social crisis. This results in a proliferation of wastewater collections which constitute potential breeding sites for Culex mosquitoes, which preferentially breed and adapt in permanent and/or semipermanent, organically polluted water collections (Nchoutpouen et al., 2019).

Currently, integrated vectors management is the most recommended strategy for the control of mosquito vectors. Larval source management (LSM) which requires better understanding of the ecology and composition of the local mosquito fauna is an important component of vector control (Mo’awia et al., 2020). Hence, characterization of mosquito larval habitats is a prerequisite to designing any efficient control strategy. To date, little work has been conducted in western Cameroon on Culex species and their larval habitats.

The current study was carried out to identify and characterize Culex mosquitoes breeding places and to determine their population dynamics in the western highlands (Dschang) and in the “Mbô” plain (Santchou). The cliff that separates Dschang and Santchou would be an example of an isolation barrier that would make it possible to assess the evolution of the specific richness of mosquitoes within the same region at different altitudes.

Methods

Study sites

This study was carried out from November 2019 to June 2020 (except in May) in two localities of the Menoua Division in the West Region of Cameroon, namely Dschang (N 05° 26.522, E 010° 03.153) in the western highlands (located at 1400 m of altitude) and Santchou (N 05° 16.945, E 009° 58.647) in the Mbô plain (located at 700 m of altitude) (Fig. 1), both separated by a forest cliff. Additional differences between the two sites are depicted in Table 1.

Fig. 1
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Map showing the study sites Dschang and Santchou with the surveyed breeding sites

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Table 1 Main characteristics of the study sites (PDCD, 2011; PDCS, 2015)
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Water collections, large or small, artificial or natural, favorable to the development of immature stages of mosquitoes (i.e., eggs, larvae, pupae) were surveyed monthly. Only water collections with Culex larvae were considered. Culex breeding sites were georeferenced using a Garmin eTrex 10 Handheld GPS (Global Position System) and revisited monthly for larval collection during both rainy and dry seasons to assess the effect of seasons on mosquito presence and distribution (Fig. 1).

Physical characterization of breeding sites and collection of Culex immature stages

Mosquito breeding sites were characterized according to seven criteria: (a) type of breeding sites (ponds, tires, swamps, streams, etc.), (b) vegetation (absent/present), (c) distance to the nearest human habitation, (d) water quality (clear or trouble), (e) depth of breeding site, (f) water movement (current/stagnant), (g) presence/absence of organic matter, and (h) presence/absence of other Culicidae larvae. Eggs, larvae and nymphs of Culex mosquitoes were collected in each breeding site of each locality using the “Dipping” technique (Service, 1993). The number of dips varied according to the size of the breeding site, the quantity of water and the presence of larvae. The collected immature stages were stored in several plastic bottles (of at least 1 L of volume) labeled and then transported to the VBID-RUBAE (Vector Borne Diseases Laboratory of the Research Unit of Biology and Applied Ecology) of the University of Dschang. They were reared to adult stage in the insectary under standard laboratory conditions (27–28°C temperature; 70–80% hygrometry) before identification.

Rearing of immature stages and identification of adult mosquitoes

Immature stages collected at the different sampling sites were transferred to labeled rearing trays. Larvae were fed with MosquiFood® as described by Tchuinkam et al. (2011), and nymphs were introduced into cardboard boxes covered with mosquito nets until adult emergence. Adult mosquitoes were later knocked down in the refrigerator (for about 15 min) and identified under a binocular magnifying glass using identification keys of Jupp (1996) and Edwards (1941).

Data analysis

Statistical analyses were performed using R software version 4.0.3 (R version 4.0.3 2020). A Mann–Whitney test was used to compare the abundance and richness of Culex mosquitoes across localities and seasons. Culex abundance according to water quality, presence/absence of vegetation, water movement, presence/absence of organic matter and presence/absence of other Culicidae larvae were assessed using a Mann–Whitney test. A Kruskal–Wallis test was used to assess Culex abundance according to sunshine (sunny, semi-shaded, shady). Spearman correlation was used to compare the abundance of Culex according to the depth of breeding site and the distance of the breeding site from human dwellings. For all analyses, the differences were considered significant at p < 0.05.

Results

Typology and distribution of breeding sites in Dschang and Santchou

A total of 44 breeding sites were identified and characterized during the field survey, with 24 sites found in Dschang and 20 in Santchou. They were grouped into seven categories: gutters (A), pools (B), tires (C), metallic containers (D), plastic containers (E and G), swamps (F) and wells (H) (Fig. 2). Overall, gutters were the most frequent breeding habitats (43.2%) followed by tires (27.3%). More specifically, in Dschang, tires and gutters were the most prevalent breeding sites (37.5% and 33.3%, respectively), while in Santchou the most encountered breeding sites were gutters and pools (55% and 20%, respectively). Wells were less prevalent in both sites (4.2% in Dschang and 5% in Santchou) (Table 2).

Fig. 2
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Breeding sites found in Dschang and Santchou

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Table 2 Distribution of breeding sites in Dschang and Santchou
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Monthly distribution of breeding sites in Dschang and Santchou

Among the seven categories of breeding sites identified, the greatest number (six breeding sites) was recorded in November (at the end of the rainy season) in Dschang (Fig. 3). The months of January and February (long dry season) recorded only one type of breeding habitats (gutters), while the months of December, March and June (rainy season) recorded two types of breeding habitats (Fig. 3). Gutters were the most encountered and colonized by Culex mosquitoes and were found in six months out of the seven months of collection, especially in June.

Fig. 3
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Monthly variation of Culex breeding sites in Dschang and Santchou

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An irregularity of the breeding sites according to months was observed in Santchou (Fig. 3). With the exception of the months of January and March (marked by the presence of only one type of habitats (gutters)), all other months recorded two types of breeding habitats, all different from 1 month to the next. Pools and gutters were the most colonized, especially in May and December, respectively.

Physical characteristics of Culex breeding habitats in Dschang and Santchou

Of the 24 breeding sites found in Dschang, 43.97% had only Culex larvae, while 56.02% were considered as mixed (presence of other larvae of Culicidae) (Table 3). On the other hand, sunny habitats contained a much greater abundance of mosquitoes (51.68%) compared to the semi-shaded and shaded habitats. In addition, mosquitoes preferred stagnant water (97.76%), without vegetation (75.28%) with a depth greater than 10 cm (72.08%). However, no significant difference of the abundance according to the different physical characteristics was observed (p > 0.05).

Table 3 Variation of Culex mosquito abundance according to the physical characteristics of breeding habitats in Dschang
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Of the 20 breeding sites found in Santchou, 46.21% consisted solely of Culex larvae, while 53.78% contained larvae of other Culicidae. Sunny breeding sites (66.04%) represented the most suitable places for Culex larvae development and appeared trouble (69.37%) with vegetation (60.23%) (Table 4). However, no significant difference of the abundance according to the different physical characteristics was observed (p > 0.05), except for the depth which significantly influenced mosquito abundance (p = 0.04575) in Santchou.

Table 4 Variation of Culex mosquito abundance according to the physical characteristics of breeding habitats in Santchou
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Abundance and richness of Culex mosquitoes in Dschang and Santchou

A total of 2706 mosquitoes belonging to four genera (Aedes, Anopheles, Culex and Lutzia) and two subfamilies (Anophelinae and Culicinae) were collected in the two localities. Culex genus was the most abundant and represented 90.4% of the total mosquitoes collected, while Aedes spp., Anopheles spp. and Lutzia tigripes accounted for less than 10% of the mosquitoes collected. Species of the Culex pipiens s.l. were the most prevalent, representing 61.79% of the total samples followed by Culex duttoni (23.17%) and Culex (Culiciomyia) sp. (05.46%). Culex mosquito abundance was higher in Santchou (n = 1290–52.72%) compared to Dschang (n = 1157–47.28%), while species richness and diversity were higher in Dschang (S = 3; H = 0.87, D = 0.54) compared to Santchou (S = 2; H = 0.23, D = 0.11) (Table 5). However, no significant difference in the abundance according to the sites was observed (p = 0.8048). This was not the case with species richness which significantly varied across sites (p = 0.0027).

Table 5 Abundance and diversity of Culex mosquitoes in Dschang and Santchou
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Figure 4 allows to appreciate species richness in both localities. It appears that the city of Dschang was richer in Culex mosquito species compared to Santchou.

Fig. 4
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Culex rarefaction curve in Dschang and Santchou

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Mosquito abundance in Dschang was higher during the rainy season (n = 643) compared to the dry season (n = 514), while the same species richness (S = 3) was observed in both seasons (Fig. 5). However, no significant difference in the abundance (p = 1) and species richness (p = 0.4232) across seasons was observed in Dschang.

Fig. 5
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Abundance and species richness of Culex per season in Dschang and Santchou

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In Santchou, the abundance of Culex was higher in the dry (n = 694) than in the rainy season (n = 596). Species richness was also greater during the dry season (S = 2) compared to the rainy season (S = 1) (Fig. 5). However, no significant difference in the abundance (p = 1) and species richness (p = 0.4232) across seasons was observed.

Discussion

The findings of this study revealed that artificial (man-made) breeding sites such as gutters and tires were the most abundant and frequent in both localities. The abundance of these two types of larval habitats could be explained by intensive human activities (economic and industrial), a poorly controlled urbanization and lack of sanitation around habitations in these localities. These results are in accordance with those obtained by Koumba et al. (2018) in agricultural areas in Gabon, Mbida et al. (2018) in the surroundings of Wouri in the Cameroon coast, and Benhissen et al. (2017) in the region of Biskra in Algeria who found that human activities and poor environmental management impact the proliferation of mosquito breeding sites. However, in a different ecosystem (the case of the forest for example), tree holes, bamboo hollows and stagnant water created by public work machines (caterpillars) represent development sites of Culex mosquitoes (Mayi et al., 2019). Additional research on the physicochemical characteristics of the different Culex breeding sites encountered should be carried out.

High densities of Culex spp. were found in turbid, stagnant and sunny water bodies, rich in organic matters in Dschang and Santchou. These results are similar to those of Berchi et al. (2012) in Algeria who indicated that Culex larvae were more frequently encountered in turbid water containing organic matter because this provides a substantial nutrient supply for their development and reduces competition rates (Souza et al., 2019). Previous studies conducted in Cameroon by Nchoutpouen et al. (2019) in Yaoundé, Ngadvou et al. (2020) in Ngaoundéré, Mbida et al. (2018) in Douala and Saotoing et al. (2014) in Maroua also revealed that mosquitoes of this genus prefer breeding sites with turbid and more or less polluted water. According to Hassaine (2002), the characters clear/turbid, sunny/shaded nature of the water in most breeding sites, added to the fact that they are poor or rich in dissolved organic matter seem to contribute largely to the proliferation of certain species. However, no significant difference in abundance based on different characteristics of immature stages was observed in Dschang and Santchou except for the depth, which significantly influenced mosquito abundance in Santchou.

Although considered as a secondary ecological factor, depth can provide an important indication on the field. It is assessed from two categories of larval habitat: deep larval habitat, in this case the depth is greater than 50 cm, and shallow larval habitat (less than 50 cm) (Hassaine, 2002). In general, the majority of Culicidae rarely frequent deep breeding sites. High densities of Culex spp. were found in shallow and sunny water bodies in Dschang and Santchou. This could be explained by the fact that, in these habitats, water temperature rises rapidly due to its exposure to sun, which accelerates the speed of larval development. In fact, eggs hatch faster when they are subjected to higher temperatures (Hassaine, 2002). Similar results on the variation in abundance according to depth were reported in An. labranchiae by Tabbabi et al. (2015).

It is known that the density of mosquito larvae in breeding sites is highly influenced by other factors such as vegetation and the surrounding habitats. In Dschang, the highest density (75%) of Culex larvae was found in breeding sites without vegetation and this is because the presence of such vegetation in water body may cause barriers for the oviposition of gravid females, shadowing the water surface as well as microbial growth. It is also very important to mention that 52% of mosquitoes in Dschang were found at a distance less than 10 m from human dwellings. The occurrence of larval habitats with a high density of mosquito larvae around houses might represent a risk of future transmission of Culex-borne diseases among the population of Dschang especially.

The knowledge of the seasonal abundance of mosquitoes is of importance in predicting the period of maximum risk of disease transmission and for carrying out an effective control program (Ashry et al., 2018). The high abundance recorded during the rainy season in Dschang shows that monthly mosquito productivity was strongly influenced by rainfall (this was not the case in Santchou). Indeed, rain offers more possibilities in the choice of breeding sites for different Culicidian species in terms of quality and quantity. These results corroborate those by Nchoutpouen et al. (2019) in Yaoundé where the abundance of Culex increased with rains. They are also similar to those of Saotoing et al. (2014) in Maroua. According to these authors, mosquitoes are present throughout the year with a greater abundance at the start and the end of the rainy season. However, in order to better appreciate mosquito dynamics across seasons, an extension of the collection period throughout the year is important.

A greater abundance of mosquitoes was observed in Santchou compared to Dschang. It is known that areas of low altitude are generally subjected to higher temperatures than those of high altitudes. However, temperature and precipitation have a direct influence on the biology of vectors and the pathogens they transmit (Carnevale & Robert, 2009). In fact, when temperature rises, female mosquitoes digest the blood meal more quickly and therefore lay eggs more frequently, which increases the chances of encountering a high density of larvae in the breeding sites. In addition, this can increase water temperature which favors larval development (Hassaine, 2002). The town of Santchou, located at a low altitude, would therefore offer more possibilities for egg-laying by female mosquitoes compared to the town of Dschang. On the other hand, this result could also be explained by the fact that in some breeding sites, the amount of water collected was greater than in others, and that egg rafts were also collected in addition to the larvae.

Overall, species richness was low because only three Culex species were identified, namely Culex pipiens s.l, Culex duttoni and Culex (Culiciomyia) spp. This could be explained by the use of only one collection method (the “dipping” or ladle stroke). Indeed, several collection methods are important to increase the chances of having greater diversity. Carlson et al. (2015) showed that at least five collection methods are necessary to have a great diversity of mosquitoes in different habitats. Dschang was more diverse than Santchou in terms of mosquito richness. Culex pipiens s.l. and Culex (Culiciomyia) sp. were found in both localities, while Cx. duttoni was only found in Dschang. The absence of Culex duttoni in Santchou could be explained by the fact that the breeding sites of Santchou did not present all the conditions required and favorable for their development. Based on studies by Mayi et al. (2019, 2020) and Nchoutpouen et al. (2019), the presence of Culicinae species and their abundance could also vary depending on the type of landscape, altitude and/or the presence/absence of vegetation cover. Indeed, areas found at high altitude generally have a greater species richness than those found at low altitude (Stevens, 1992). These results are contrary with the work by Tchuinkam et al. (2010) on the bionomic of Anopheles following an altitudinal transect in the region of West Cameroon, where the diversity of Anopheles species decreased from areas of low altitudes to those of high altitudes. A similar study at a much higher altitude and on the forest cliff itself should be carried out in order to assess the evolution of Culex mosquitoes following an altitudinal transect in the West Region of Cameroon.

Conclusions

A great diversity of Culex breeding sites were encountered in both localities of which mostly were shallow, close to human dwellings, sunny, trouble, loaded with organic matter and of an anthropogenic nature. While Santchou recorded the greatest abundance of Culex, Dschang was found to be more diverse in terms of species richness, suggesting a possible effect of the altitudinal differences of the two localities on Culex mosquitoes. The proliferation of Culex mosquitoes in the two localities would be attributable to the unsanitary conditions and to human activities which create and ensure the maintenance of breeding sites for mosquitoes; this could increase the risk of vector-borne diseases to the human population of Dschang and Santchou. A sustainable public health campaign on vector management and control should therefore be intensified in these cities.