Anopheles leesoni Evans 1931, a Member of the Anopheles funestus Group, Is a Potential Malaria Vector in Cameroon

Background: Understanding the biology of Anopheles malaria vector species is essential to planning effective and sustainable malaria control strategies in endemic countries. This study reported the implication of Anopheles leesoni in malaria transmission in Cameroon, Central Africa. Methods: Mosquitoes were collected in three localities from May 2015 to March 2018 using electric aspirators and Centers for Disease Control light traps (CDC-LT). Anopheles funestus sensu lato (s.l.) mosquitoes were identified as species using polymerase chain reaction assay (PCR). Furthermore, Plasmodium falciparum infection status was determined using the enzyme-linked immunosorbent assay (ELISA) leesoni was found positive for P. falciparum (infection rate: 10.98%) in Cameroon. Conclusion: A very high P. falciparum infection rate was observed in this study in A. funestus s.s., highlighting its high implication in malaria transmission in Cameroon. Furthermore, the detection of P. falciparum infection in A. leesoni calls for more attention towards this neglected vector species.


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. 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.

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.

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.

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

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.

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.

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%).

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

Plasmodium Infection Rates
Plasmodium falciparum infection rates are given in

No difference in terms of infection was observed between both species in
Yaoundé (P = 0.11).

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.