Background
Despite several decades of control efforts, malaria remains a major public health problem in most tropical and subtropical regions of the world, especially in Africa [
1]. The uncontrolled population growth in many areas has led to extensive deforestation, irrigation, and unplanned urbanization. These high populations and associated environmental modifications create ecological conditions favouring the proliferation of arthropod vectors, including malaria-transmitting mosquitoes [
2]. Vector control, mainly based on the extensive use of Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS), is a corner stone in current malaria control and elimination strategies [
1]. With adequate knowledge of larval habitats, these two weapons can be complemented effectively with larval source management under a specific set of environmental conditions [
3].
In Cameroon, malaria annually accounts for 35–40 % of deaths in health facilities, 40–45 % of out-patient consultations, and 30 % of hospitalisations [
4]. It is also responsible for 26 % of job and school absenteeism and over 40 % of domestic health expenses [
5]. Malaria infection is essentially due to
Plasmodium falciparum, followed by
P. ovale and
P. malariae [
6]
. Seven anopheline species play major roles in
Plasmodium parasite transmission, among which are
Anopheles (An.) arabiensis,
An. gambiae and
An. coluzzii, three sibling species of the
An. gambiae complex [
7].
Anopheles arabiensis is mostly found in the Northern savannah Regions, while
An. gambiae s.s. and
An. coluzzii are widespread throughout Cameroon [
8]. Although
An. gambiae s.s. and
An. coluzzii share the same resources such as vertebrate hosts or freshwater habitats, they have been shown to diverge in some of their biological and ecological requirements [
9‐
11]. In the dry savannahs of West Africa,
An. gambiae s.s. preferentially breeds in temporary aquatic habitats and is found during the rainy season only, whereas
An. coluzzii is present all year round, breeding in man-made permanent aquatic habitats [
10,
11]. Species are sharply segregated along a gradient ranging from “permanent-anthropic” habitats, exploited by
An. coluzzii to “temporary natural” habitats where
An. gambiae s.s. and
An. arabiensis are found [
12]. Consequently, larval ecology of this species complex appears clearly linked to habitat hydro-periodicity and to the ecological communities they support in relation to anthropogenic activities. In Cameroon, little is known about habitat segregation between
An. gambiae s.s. and
An. coluzzii in Island areas.
In the framework of malaria elimination, large-scale vector control interventions based on wide use of LLINs are being implemented by the National Malaria Control Programme (NMCP) in Cameroon [
13]. Indeed, a nationwide free distribution of eight and a half million LLINs was carried out in 2011 by the NMCP, and a second nationwide distribution of twelve million LLINs is ongoing since 2015. However, several studies have reported insecticide resistance in
An. gambiae s.s., An. arabiensis and
An. coluzzii in the Northern, Central, Southern, Eastern, Western and Coastal Regions of Cameroon [
14‐
16]. The resistance to DDT and pyrethroid insecticides reported in species of the
An. gambiae complex has been conferred by elevated activity of esterases, glutathione s-transferases, P450 oxidases, and
kdr L1014F or L1014S mutations, with some populations displaying multiple resistance mechanisms [
17‐
19]. The ongoing selection and spread of insecticide resistance in Cameroon is seen as a significant threat impeding progress towards malaria elimination in the country. To develop a comprehensive resistance management plan, it is essential to understand the spatio-temporal distribution of insecticide resistance across the country, and to continuously monitor resistance emergence in representative ecological areas. These data would be used to guide the choice of insecticides for effective malaria vector control. Until recently, the malaria entomological profile in the Manoka island was unknown, despite the potential of this setting as an experimental site for malaria elimination trials, due to its small size, insular position and manageable connections with the mainland. The current study was aimed at filling the knowledge gap on malaria vector larval habitats and insecticide resistance in the Manoka island and Youpwe crossroad quarter in mainland Douala. We report here the occurrence of permanent active larval habitats and multiple insecticide resistance mechanisms in the two
An. coluzzii populations, including at least the
kdr 1014 F mutation and P450 oxidase-based mechanisms.
Discussion
Irrespective of the period of survey, three types of permanent or semi-permanent breeding sites for An. coluzzii were commonly recorded among the most active larval habitats in the Manoka rural island area and the Youpwe suburban area. These were drains, ponds and boats. More interestingly, at least a quarter of the active water bodies in both localities were boat-breeding sites, highlighting a previously unreported important niche for An. coluzzii breeding in these areas.
Although Tene-Fossog and colleagues [
38] predicted a predominance of
An. coluzzii along the coastline of west and central Africa, there was still limited information on
An. coluzzii occurrence and larval habitat on the Manoka island area. Small, temporary sunny water bodies, relatively clean and mostly without overhanging vegetation have been recognized by several studies as preferred breeding habitats for
An. gambiae s.l. [
39]. Other studies have reported the breeding of
An. gambiae s.l. in large permanent and polluted water bodies [
40,
41]. More precisely, ecological niches of species of the
An. gambiae complex were found to segregate according to habitat type and temporality [
42]. In Cape Coast (Ghana),
An. coluzzii was reported to actively breed in diverse habitats, including footprints, tyre tracks, rain pools during the rainy season, and choke gutters and other organic polluted habitats during the dry season [
43]. Although temporary breeding sites may be equally important in the ecology of
An. coluzzii, they were not very active during the survey periods in Manoka and Youpwe. We therefore focused the current study on permanent and semi-permanent breeding sites which were positive during the two larvae collection surveys. The positivity of permanent and semi-permanent breeding sites for
An. coluzzi in the current study was consistent with Gimonneau et al. [
12], who reported that “permanent-anthropic” habitats are mostly exploited by
An. coluzzii (former M form), while “temporary natural” habitats are colonised by
An. gambiae s.s (former S form) and
An. arabiensis. Furthermore, the exclusive identification of
An. coluzzii in the current study supports previous reports of this mosquito species representing the dominant malaria vector species in the Littoral Region of Cameroon [
11].
On the other hand, interbreeding between mosquito populations may influence their dispersal and the subsequent evolution of insecticide resistance. The present study revealed insecticide resistance in two interbreeding
An. coluzzii vector populations, and highlighted seasonal variations in resistance patterns (wet/dry). Similar variations in susceptibility of
An. gambiae s.l. populations to insecticides were reported in some African countries [
44‐
47] and North Cameroon as well [
48]. They were linked to seasonal immigration of
kdr-resistant
An. gambiae s.s. mosquitoes originating from the neighbouring cotton fields, temporal variations in species composition within the
An. gambiae s.l. complex, or cycles of insecticide treatments for agriculture and/or public health purposes. In Manoka and Youpwe, seasonal variations of resistance levels over the year might result from local fluctuations of vector population dynamics according to rainy events and human activities related to agriculture and household protection against mosquito bites (e.g. use of coils, mats, etc. purchased from the market). In addition, these variations may be related to the migration of
An. coluzzii specimens through boat transportation between the two areas, and to a lesser extent to changes in species composition, since
An. gambiae s.s. has been reported in some quarters in Douala.
Furthermore, the rapid scale-up of insecticide-based malaria control measures in Africa during the first decade of the 2000s has had measurable effects on malaria vectors, especially on
An. gambiae and
An. coluzzii, which are highly dependent on humans for blood and indoor resting sites. In some cases, selection pressure from these measures has been strong enough to drive
An. gambiae and
An. coluzzii to local extinction [
49]. In other cases, the selection pressure has resulted in rapid evolution of pyrethroid resistance and increase of
kdr L1014F allelic frequencies, as reported in Douala and Yaounde in Cameroon [
50]. The two subsequent nationwide LLINs distribution campaigns launched in 2011 and 2015, in addition to the use of insecticides in agriculture would be expected to impact on local malaria vector populations.
Mosquito samples used in the current study were collected during two periods (December 2013 and April 2014) in order to reflect seasonal variations of DDT, permethrin and deltamethrin resistance in
An. gambiae s.l. from Manoka and Youpwe. However, the low productivity and the flush of larval breeding sites during the dry and rainy seasons respectively precluded us to complete all the susceptibility tests for the three targeted insecticides, with or without PBO. Nevertheless, the 14 tests that were successfully performed provided an overview of DDT, permethrin and deltamethrin resistance phenotypes and the
kdr 1014 genotypes in
An. coluzzii populations from the study sites. The level of
An. coluzzii resistance to DDT and pyrethroids as well as the
kdr 1014 F allelic frequencies recorded in the current study are higher than those previously reported in
An. gambiae s.s and
An. coluzzii from some remote districts of the Douala city [
51]. These results testify that the spread of the
kdr alleles is an ongoing process in
An. gambiae s.l. mosquito populations from Cameroon [
18,
51,
52], as well as elsewhere in Central Africa [
53,
54]. The high frequencies of
kdr L1014F allele in surviving samples (75-88 %), with predominance of the
kdr 1014 F/1014F homozygote genotype (60-84 %), suggests a strong involvement of this allele in the resistance phenotype. The
kdr 1014 F allele being recessive, the presence of 1014 L/1014F heterozygotes (11–32 %), as well as a few 1014 L/1014L homozygotes (<10 %) in surviving samples suggest involvement of other resistance mechanisms. Indeed, pre-exposure of mosquitoes to PBO resulted in reversion of pyrethroid resistance in both tested populations, suggesting involvement of P450 oxidases in this resistance [
55]. The synergistic effect of PBO and high frequencies of
kdr L1014F allele in surviving specimens strongly suggests multiple pyrethroid resistance mechanisms in
An. coluzzii from Manoka and Youpwe. These findings fit well in the overall picture presented in previous surveys in Douala and other settings in Cameroon [
19].
In West Africa,
An. coluzzii seems to have responded to insecticide selection pressure by co-opting pyrethroid and DDT resistance in the form of the L1014F mutation from
An. gambiae through adaptive introgression [
56]. The selection of the
kdr 1014 F allele is likely stronger in
An. coluzzii than in sympatric
An. gambiae, owing to either (i) the need for the resistance allele in the presence of insecticide to overcome the selective disadvantage of
An. gambiae or (ii) phenotypic penetrance of L1014F differing in the
An. gambiae genetic background [
57]. In the Bioko Island (Equatorial Guinea, neighbor to South Cameroon), a high and unusual distribution of
kdr L1014F frequencies in
An. coluzzii (former M molecular form of
An. gambiae s.s.) was also reported [
58]. In some quarters in Douala, both
kdr L1014S and L1014F alleles have been reported in
An. coluzzii specimens surviving susceptibility tests, at frequencies of 4 % and 38 % respectively [
19]. The absence of the L1014S allele in samples from Manoka and Youpwe suggests its selective disadvantage compared with the L1014F allele in these areas. The L1014F allele may have arisen in coastal Cameroon from in situ mutation events, and evolved through gene flow or local selection pressure due to insecticide use in vector control interventions and individual protection tools against mosquito bites [
59]. Indeed, DDT was massively used for indoor residual spraying in the framework of the pilot malaria eradication programme in Douala during the 1950s [
60]. Furthermore, Desfontaines et al. [
61] reported the widespread use of insecticides for household protection against mosquito bites in Douala.
The influence of evolutionary forces (mutations, gene flow and selection) in shaping An. coluzzii populations from Youpwe and Manoka might therefore be counterbalanced by ecological fitness costs, resulting in the development of insecticide resistance.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JE, AMM, CEEM and LGL conceived and designed the study protocol. AMM, PNA, AT, WEE, DT, RT, JB, PAA and JE carried out field and laboratory assays. JE, AMM, JB and PA analysed and interpreted data, and wrote the paper. LM, RM, PNA, CEEM and LGL critically reviewed the manuscript. All the authors read and approved the final version of the manuscript.