1. Introduction
In Africa, the most important and widespread vectors of malaria belong to the An. gambiae complex and the A. funestus group [1] [2] [3]. Adult members of these two complexes/groups are difficult to distinguish morphologically [4] [5], necessitating molecular techniques for accurate identification [1] [6] [7].
Within the A. funestus group, A. funestus s.s. is the only member that plays a significant role in the transmission of human malaria throughout the African continent, but other species of A. funestus group have been naturally found infected with P.falciparum [8]. This mosquito is widely distributed throughout tropical Africa and its breeding site is permanent or semi-permanent. Its activity extends even during the dry season where other malaria mosquito vectors, such as A. gambiae s.l. are usually less abundant [9]. As for other members of the A. funestus group, some reports indicated that A. rivulorum may be involved in malaria transmission in some situations [8] [10] and P. falciparum has been reported from A. parensis and A. leesoni [11]. A. vaneedeni has been experimentally infected with P. falciparum [12] which has recently been isolated in natural populations of this species in South Africa [13]. No reports of any involvement in malaria transmission for the remaining members of the A. funestus group were found. Despite their morphological similarity, the species of A.funestus group shows different vectorial capacities and then different malaria transmission capacities. Therefore, there is a necessity to determine the predilection place of action of each species in order to readapt malaria vector control decisions and operations, focusing on really affected areas and making vital commitments in all African countries where financial resources relating to related malaria control are limited.
Historical evidence suggests that in order to conduct an efficient vector control program, there is a necessity to identify and distinguish vector species from non-vector species. Control measures against A. funestus s.s., which is an anthropophilic and endophilic vector, favour exophilic members of the A. funestus group, increasing their density [14]. For example, in South Africa [12] [15], Kenya [16], and Tanzania [14] [17], indoor spraying used to eliminate A. funestus s.s. was followed by an upsurge of “funestus look-alike” specimens, contributing to the failure of the control program. However, further investigation revealed that these mosquitoes belong to A. vaneedeni, A. parensis, A. rivolurum, or A. leesoni. Each of them is occasionally or rarely implicated in the transmission of malaria to humans, and their zoophilic and exophilic habits probably reduce exposure to insecticides.
Reliable species identification is indeed important to assess the relative role played by each species in the transmission of Plasmodium and improve our ability to evaluate the efficacy of vector control measures implemented in areas where several species of the A. funestus group are present. In the past, species identification has mainly been performed using either morphological or cytogenetic methods. However, the development of PCR-based methods has greatly facilitated the identification of species in the group [7] [18]. It is commonly asserted that malaria transmission in Africa is maintained by members of the A. gambiae complex [19]. However, in several parts of the continent, other mosquito species contribute to the transmission of the parasite, including A. funestus s.l. and A. nili. Previous studies in Cameroon defend that A. funestus s.s. is the main, if not the only vector of the A. funestus group responsible for transmission of malaria parasites [7] [20] [21] [22]. Although A. leesoni [7] [20] [21] have been found in several malaria foci, their role in the transmission of malaria in Cameroon has not been further studied.
In this paper, we provide evidence incriminating A. leesoni in the transmission of malaria in Cameroon. Demonstrating at the same time the presence of two species of the A. funestus group in this country, where malaria transmission is a serious public health problem.
2. Methods
2.1. Study Area
The study was carried out in three sites belonging to the forest and savannah domains of Cameroon (Figure 1).
Mebelong (6˚46'N, 11˚70'E) is located in the Adamawa region, approximately 350 km from Yaoundé, the capital city of Cameroon. The village is situated at the vicinity of a lake that represents a potential breeding site for A.funestus s.s. mosquitoes throughout the year [23]. The climate is Sudano-Guinean characterized by an eight-months rainy season from March to October, and a dry season of four months extending from November to February [24].
Obout (12˚53'N, 35˚7'E) and Yaoundé (3˚52'N, 11˚27'E) are located about 30 km apart within the forest regions area of the Centre region. The climate is alike to that of Equatorial Guinea, characterized by two rainy seasons extending from August to October, and from April to June. There are also two dry seasons running from November to April and from June to July [24]. The village Obout is situated in rural zone and is surrounded by an evergreen forest. Within the village, there are several fish ponds bordered with emergent vegetation suitable for the development of Anopheles mosquito larvae, particularly those of A.funestus group.
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Figure 1. Species composition within the A. funestus group in the three localities (N: number of mosquitoes identified by PCR).
The town Yaoundé is made up of wetlands and a degraded forest surrounding the city. There are also lakes and fish ponds suitable for the development of anopheline mosquitoes. Yaoundé features an equatorial climate with two rainy seasons extending from March to June and from September to November lasting 7 to 8 months [24].
2.2. Ethical Consideration
The study was approved by the Cameroonian national ethical committee for research on human health (statement N˚ 2015/01/535/CE/CNRERSH/SP). Verbal informed consent was obtained from each head household before the team entered their houses for mosquito collection.
2.3. Mosquito Collections and Identification
Mosquitoes were collected from May 2015 to December 2017 in Obout and Mebelong and from May 2017 to March 2018 in Yaoundé.
In Obout and Mebelong, indoor resting mosquitoes were collected in human dwellings in the morning 10 to 15 houses, between 7:00 and 10:00 AM using electric aspirators (Rule In-Line Blowers, Model 240) whereas in Yaoundé mosquitoes were collected from 6:00 PM to 6:00 AM using CDC light Traps placed indoors and outdoors in 10 to 15 houses. After species identification using morphological keys [4] [5], only mosquitoes belonging to A. funestus group were included in the subsequent analysis.
2.4. Molecular Identification of Anopheles funestus Members
Molecular identification of specimens of A. funestus group was performed following the species-specific protocols described by [21] and [7]. Abdomen, legs and wings were used for genomic DNA extraction as described previously [25]. The primers contained in Table 1 were used. A final 25 μL reaction volume of PCR contained 2.5 μL of 10× buffer including 15 mM MgCl2, 5 pmol of each primer, 200 μM of each dNTP, and 0.5 units of Taq polymerase unit. Amplification started with an initial denaturation step at 94˚C for two minutes, followed by 36 cycles of denaturation at 94˚C for 30 seconds, annealing at 45˚C for 30 seconds, and elongation at 72˚C for 40 seconds, with a final extension step at 72˚C for five minutes. The PCR products were loaded and visualized on regular 1.5% agarose gels stained with ethidium bromide.
2.5. Detection of Plasmodium falciparum Infection
Head and thorax of each female mosquito were subjected to indirect enzyme-linked immunosorbent assay (ELISA) for the presence of P. falciparum circumsporozoite protein (CSP) using monoclonal antibodies 2A10 as described by Wirtz et al. [26]. One positive control and ten negative controls were added to each microtitre plate. Negative controls were head and thorax of unfed A. gambiae s.s. (Kisumu) from laboratory colonies maintained at OCEAC. Absorbance was measured at 405 nm using a microtitre plate reader (BioTek ELx800, Swindon, UK). The cut-off value for positive specimens was estimated at twice the mean value of the negative controls.
2.6. Statistical Analysis
Infection rates were determined as the percentage of mosquito species samples found positive for P. falciparum over the total number of specimens tested.
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Table 1. Sequences of primers used for molecular identification of An.funestus species.
3. Results
3.1. Anopheles Mosquito Population
A total of 12,744 resting Anopheles mosquitoes were collected during the study period including 7857 A.gambiae s.l. and 4887 A.funestus s.l. (Table 2). Anopheles funestus s.l. was by far the most abundant in Obout and Mebelong representing 74.42% and 97.12% of the total Anopheles mosquitoes caught respectively. By contrast, A.gambiae s.l. was the most frequent species in the city of Yaoundé (91.46%).
3.2. Anopheles leesoni Abundance and Distribution
The Anopheles funestus group, as revealed by the molecular identification of 1389 individual mosquitoes, was composed of two species, including 1307 A.funestus s.s. (94.10%) and 82 A.leesoni (5.90%). Both species were found in all three localities (Figure 1). However, the proportion of A.leesoni was higher in Yaoundé (57/390: 14.62%) than in Obout (16/474: 3.38%) and in Mebelong (9/525: 1.71%).
3.3. Plasmodium Infection Rates
Plasmodium falciparum infection rates are given in Table 3. Of a total of 1389 head and thorax analyses, 284 were positive, corresponding to a high global circumsporozoite rate of 20.45%. Among the mosquitoes tested, 21.04% (275/1307) were infected with A. funestus s.s. and 10.98% (9/82) for A. leesoni. Although the infection rate of A. funestus s.s. appeared higher in Obout and Mebelong compared to A. leesoni, this difference was not statistically significant (P > 0.05). No difference in terms of infection was observed between both species in Yaoundé (P = 0.11).
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Table 2. Number of A.funestus s.l. and A.gambiae s.l. mosquitoes collected in Obout, Mebelong and Yaoundé.
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Table 3. Circumsporozoite protein (CSP) rates of A.funestus s.s. and A.leesoni mosquitoes from the study locations.
4. Discussion
The control of Anopheles vector populations is the pillar of malaria elimination strategies. Identifying primary disease vectors and understanding their biology and geographic distribution is crucial to plan efficient control strategies. For several decades attention has been focused of mosquitoes from A. gambiae complex which have for a long time been considered as the most efficient malaria vector throughout Africa continent. However, the recent increase of interest in other Anopheles species, such as those from Anopheles funestus group led to the change of this paradigm. Similar to this study A. funestus s.s. was repeatedly reported to be widespread and highly infected with P. falciparum, thus playing a major role in malaria transmission in East, Central and West Africa.
If knowledge and control of major vector species using insecticide and insecticide-treated tools successfully contributed to malaria reduction over the past decade [27], the real challenge for malaria elimination and eradication could arise from secondary vectors that sustain residual malaria transmission in the absence of primary vectors. Unfortunately, little is known regarding the distribution and biology of such secondary vectors.
In this study, we have demonstrated that A. leesoni is sympatric with A. funestus s.s. in forest and humid savannah ecosystems in Cameroon and was infected with P.falciparum in all the study sites. Previous studies have already reported the presence of A. leesoni in Yaoundé but not in Obout and Mebelong. However, to our knowledge, this is the first time this species has been incriminated as a malaria vector in Cameroon [28]. In other regions of the continent, there are some reports [11] of the possible carriage of P. falciparum parasites by A. leesoni, but there is no or little evidence of its role as a secondary malaria vector. Other members of the A. funestus groups, such as A. rivulorum and A. vaneedeni, were also found infected by malaria parasites in laboratories and in nature. Anopheles rivulorum has been implicated in malaria transmission or found to harbour P. falciparum parasites in Kenya [10], Tanzania [8] [11] and Zambia [29]. Anopheles vaneedeni has been experimentally infected with Plasmodium in the laboratory [12] and was recently found infected in nature [13].
Although we didn’t assess A. leesoni’s feeding behaviour by determining the origin of the blood meal in the abdomens of mosquitoes, the fact that blood-fed A. leesoni was found resting inside human dwellings suggests that this species is endophilic and anthropophilic. Previous research by Temu et al. [11] in Tanzania revealed a preference for humans (81.8%) over goats (0%), with the species also resting inside human dwellings in Kenya [29] and West Africa [30].
Relatively high rates of infections of P. falciparum were detected in A. funestus s.s. and A. leesoni collected in our study area. This is the first report on Plasmodium infection in A. leesoni in Cameroon. Although this vector has been reported from different sites in East Africa and has been shown to play a major role in malaria transmission in Africa, no information is so far available on its infection with malaria parasites in Central Africa (Cameroon). Such high infection rates in non-vectors should be interpreted with caution because none of the previous studies have reported any samples of A. leesoni infected with the Plasmodium parasite, either by salivary gland dissections or ELISA detection methods [4] [21]. While we cannot exclude the possibility that the ELISA is detecting sporozoites in the salivary glands alone, it is likely that the method is also picking up parasites in the thoracic hemocele. Therefore, unlike salivary gland dissections, ELISA does not guarantee that the mosquito is infectious unless it is carried out on the salivary glands themselves. Further studies are required involving detection of parasites by ELISA or PCR performed on salivary glands dissected from wild members of the A. funestus group.
Although it is in low abundance in this study, A. leesoni appears to transmit malaria as well as A. funestus s.s. in Cameroon. As reported in Tanzania [11], more than one species within the A. funestus group was found infected with P. falciparum, therefore sustaining the need to identify and adjust the list of malaria vectors that belong to species groups or complexes in order to establish areas of sympatric existence and to assess the role played by each species in malaria transmission. This information will improve our ability to evaluate the efficient and strategic planning of vector control measures.
In conclusion, since mosquito abundance displays temporal and spatial fluctuation and since more than one species within the A. funestus group was found infected with P. falciparum, it is important to characterize the spatial distribution of the A. funestus s.s., A. rivulorum, and A. leesoni according to the malaria endemicity rate. Among members of the A. funestus group, the species composition and species diversity are likely to differ locally, leading to significant impact on efficiency of malaria vector control management.
Acknowledgements
This work was supported by a Wellcome Trust Training Fellowship in Public Health and Tropical Medicine (WT 102543/Z/13/Z) to CN and a Wellcome Trust Fellowship Senior in Public Health and Tropical Medicine (WT 202687/Z/16/Z) to CAN.
Authors’ contributions
CAN and CN conceived and designed the study protocol; EK, LDD and LN participated in data collection and laboratory analyses; CAN, CN, PAA, FN and CSW critically revised the manuscript; EK, CN and CAN interpreted, analysed data, and wrote the manuscript. All authors read and approved the final manuscript.
Abbreviations
CDC: Centre for Disease Control and Prevention; ELISA: Enzyme Linked Immunosorbent Assay; PCR: Polymerase Chain Reaction; s.l.: sensu lato; s.s.: sensu stricto; A.: Anopheles