Role of Anopheles (Cellia) rufipes (Gough, 1910) and other local anophelines in human malaria transmission in the northern savannah of Cameroon: a cross-sectional survey

The discovery at the turn of the nineteenth century that malaria was transmitted by mosquitoes initiated an outburst of interest in the description and implication of various species in malaria transmission [1]. Human plasmodial species are transmitted by mosquitoes of the genus Anopheles, which includes 465 formally recognised species [2]. Amongst these, over 140 species have been identified in Africa [3, 4], of which less than 20 have been shown to support the development and propagation of human Plasmodium species [5]. However, their relative contribution to malaria transmission greatly varies depending on their behaviour (host seeking, feeding and resting) and densities as influenced by environmental conditions. These differences in Anopheles behaviour and density, along with vector longevity, are key factors driving malaria transmission and epidemiological patterns observed across Africa [6, 7]. Therefore, in areas with numerous potential vectors, an in-depth understanding of vectors dynamics is fundamental to designing interventions tailored to the local eco-epidemiological situation [8, 9].

The malaria vectorial system in Cameroon is very complex. To date, over 50 Anopheles species have been described which are heterogeneously distributed across the eco-epidemiological zones of the country. Fifteen species have been demonstrated to support the development and spread of human Plasmodium spp.: An. gambiae Giles, 1902; An. funestus Giles, 1900; An. arabiensis Patton, 1905; An. nili (Theobald, 1904) and An. moucheti Evans, 1925 are classified as major vectors [10]. The other species amongst which are An. pharoensis and An. paludis play only a minor localised secondary role in transmission [11–14], and they may help to augment or extend the malaria transmission period [15, 16]. For the first time we report the importance of An. rufipes (Gough, 1910), as vector of human Plasmodium in Cameroon. Although a few studies have attempted to associate An. rufipes with human malaria transmission [17, 18], this species has generally been reported to be zoophilic [3, 4] with high densities in rice-growing areas [19, 20]. Even though this species has often been collected landing on human volunteers in several studies in Cameroon [21–23], its role in malaria transmission has never been investigated.

The identification and incrimination of vectors in malaria transmission as well as their relative contribution to transmission is critical for the implementation of an efficient malaria control strategy. Some control programmes like the Garki project, failed because of the erroneous identification of the vectors involved in malaria transmission, and insecticide resistance in An. arabiensis in Nigeria [35, 36]. To date, malariologists have paid little or no attention in most areas to An. rufipes as this species has generally exhibited high zoophilic tendencies. Elsewhere, the abundance of this species was found to peak particularly at the end of the rainy season in rice-growing areas, with very short periods of malaria outbreak resulting [17–20]. Anopheles rufipes breed commonly in small collections of water such as residual puddles in river beds, hoof prints of cattle, swamps and rice irrigation schemes, with the larvae staying close to the borders and among vegetation. This is important as cattle rearing and rice cultivation and irrigation are common practices in the study sites, which usually experience floods with long retention of surface water during the wet season. As part of a study to determine the impact of insecticide resistance on the effectiveness of vector control tools like the LLIN in the north of Cameroon, the observed unexpectedly high density and anthropophilic behaviour of this species incited our curiosity to investigate its role in P. falciparum transmission. For the first time in Cameroon, An. rufipes has been identified as an important local vector of P. falciparum. A few earlier studies have described this species as weakly anthropophilic [18] and potentially involved in malaria transmission [3, 37, 38]. More recently in Burkina Faso, Plasmodium was detected in An. rufipes oocyts, further implicating it as a potential vector of human malaria parasites [39]. Thus, An. rufipes may be a more important and wider spread vector than previously thought and therefore this is important for malaria elimination strategies.

In Cameroon, five major malaria vectors (An. gambiae, An. funestus, An. arabiensis, An. nili and An. moucheti) have been described [10] with nine secondary vectors of local importance (An. paludis, An. carnevalei, An. coustani, An. marshallii, An. ziemanni, An. pharoensis, An. hancocki, An. wellcomei and An. ovengensis). Anopheles rufipes is now added to this plethora of vectors, further complicating the malaria vectorial system in the country. In the past, the role of this species in malaria transmission was largely neglected due its strong zoophilic tendency which leads to claims of its lack of importance. Moreover, An. rufipes had high parous rate wherever it was collected, suggesting the propensity to survive longer and being able to maintain and even extend P. falciparum transmission beyond the normal duration.

The vast plethora of Anopheles species observed in this study could be linked to the diversity of existing biotopes that enhance the proliferation of such species within the health districts. Anopheles arabiensis, An. coluzzii and An. gambiae (s.s.) were  the An. gambiae (s.l.) siblings found with An. arabiensis being the most frequently encountered species. The relative abundance of An. gambiae (s.s.) and An. arabiensis varied across the years and by health districts with twice as many An. arabiensis. This is unlike observations by Robert et al. [40] in southern Burkina where the density of An. gambiae (s.s.) was nine-fold greater than that of An. arabiensis among residual endophilic mosquitoes. This difference however, might be due to differences in the microenvironments and the methods of collection. Both species are typical malaria vectors of the sahelian zone [41], with their densities peaking especially during the rainy season. As observed with An. rufipes in this study, species like An. pharoensis, An. funestus and An. coustani have been shown to contribute significantly to malaria transmission [23]. For An. funestus, the density remained relatively constant irrespective of the season [42]. Akin to studies conducted in Burkina Faso, An. rufipes constituted a significant proportion of the anophelines collected in this study [39]. This however differs markedly with observations from the HLC collections in the coastal areas and northern regions of Cameroon [21, 22] and Chad [43], where An. rufipes density was low. The vast number of prolific breeding sites (rice fields, river banks and pools) during the study periods may explain the high density of An. rufipes [19, 20].

With regard to the biting rates of the anophelines, except in Mayo Oulo where the highest An. rufipes densities were recorded in 2012 and 2013, An. gambiae (s.l.) generally had the highest biting rates throughout the study (Table 2). Analyses of the blood meal sources among the members of the An. gambiae complex revealed An. gambiae despite its low density compared to An. coluzzii and An. arabiensis, to be the most anthropophagic. Amongst the vectors for which the blood meal source was confirmed, they were observed to feed mainly on cow, which is the most widely domesticated animal in this part of the country. Anopheles rufipes was observed to feed on both humans and animals confirming the species opportunistic behaviour or may be simply due to the imposing environmental conditions. Livestock rearing is a common practice with animals like dog, chicken, horse, goat and donkey in addition to those analyzed. Unlike previous reports on An. funestus being the most anthropophagic Anopheles species [33, 44–47] its human blood index in this study was very low. This may be due to the presence of a variety of domestic animals that provide an alternative source for blood-feeding or due to the occurrence of other members of the An. funestus group of species. In fact, Anopheles funestus (s.l.) comprises  at least nine species that are morphologically identical [3]: An. funestus (s.s.), An. vaneedeni Gillies & Coetzee, An. parensis Gillies, An. aruni Sobti, An. confusus Evans & Leeson, An. rivulorum Leeson, An. fuscivenosus Leeson, An. leesoni Evans and An. brucei Service. All except An. funestus (s.s.) and to some extent An. rivulorum are purported to be exclusively zoophilic and non-vectors [48]. Despite the fact that many authors reported a high infection rate of An. funestus during dry season [3, 49], Antonio-Nkondjio et al. [22] observed a pronounced anthropohilic behaviour of An. funestus during the wet season while this species was exclusively zoophilic during the dry season. Other earlier studies [50, 51] also reported besides An. funestus (s.s.) the presence of Anopheles leesoni Evans and Anopheles rivulorum-like in tributaries of the Benoue River in the northern region of Cameroon. Based on these observations and the current HBI results, there is the likelihood of the existence of some zoophilic members of An. funestus group in this area. Therefore, it would be important that subsequent studies in this area should consider the identification of the members of the An. funestus group of species and their contribution to human malaria transmission.

On a general note, in addition to the Anopheles species displaying endophagic and exophagic tendencies, the parity rates were observably high in all HDs. Thus, despite the high LLIN coverage in the study localities [52], the malaria vectors survive long enough to sustain malaria transmission locally.

The infection rates varied enormously between the health districts. Overall, seven Anopheles species (An. arabiensis, An. coluzzii, An. gambiae, An. funestus, An. pharoensis, An. rufipes and An. paludis) were found infected with P. falciparum. Even though the CSP rates were higher than those obtained elsewhere under similar eco-epidemiological conditions [22], measures were taken to minimize false positive CS ELISA in these areas with an extensive animal reservoir flow for blood-feeding. Thus, only high absorbance readings (mean plus three standard deviations of negative controls) were considered. Nevertheless, this CSP-ELISA may overestimate the infection rate and all positive samples need to be confirmed with PCR [53]. Relatively low malaria transmission intensity was observed throughout the years in all health districts. The observed EIR estimate of 0.014 to 1.221 infective bites/person/night was consistent with other records in the sahelian zone [25]. However, the transmission intensity during the study was pronounced in the irrigated rice paddies of Pitoa, which is not uncommon to other such irrigation schemes that generate a variety of entomological and epidemiological situations with permanent water bodies that are prolific for Anopheles breeding and consequently increasing the risk of malaria in surrounding population [54].

The study highlights the epidemiological role of An. rufipes alongside the members of the Anopheles gambiae complex, and several other sympatric species in human malaria transmission during the wet season in northern Cameroon. Thus, for the first time in Cameroon, the role of An. rufipes as an important local malaria vector has been made evident. The study also portrays the need to carry out further studies to document the implication of this species and other presumably non-malaria vectors in human malaria transmission in other parts and to review and update the malaria entomological profile in the country and the Afrotropical region.