BackgroundAnthropogenic habitat disturbance is a prime cause in the current trend of the Earth’s reduction in biodiversity. Here we show that the human footprint on the Central African rainforest, which is resulting in deforestation and growth of densely populated urban agglomerates, is associated to ecological divergence and cryptic speciation leading to adaptive radiation within the major malaria mosquito Anopheles gambiae.Methodology/Principal FindingsIn southern Cameroon, the frequency of two molecular forms–M and S–among which reproductive isolation is strong but still incomplete, was correlated to an index of urbanisation extracted from remotely sensed data, expressed as the proportion of built-up surface in each sampling unit. The two forms markedly segregated along an urbanisation gradient forming a bimodal cline of ∼6-km width: the S form was exclusive to the rural habitat, whereas only the M form was present in the core of densely urbanised settings, co-occurring at times in the same polluted larval habitats of the southern house mosquito Culex quinquefasciatus–a species association that was not historically recorded before.Conclusions/SignificanceOur results indicate that when humans create novel habitats and ecological heterogeneities, they can provide evolutionary opportunities for rapid adaptive niche shifts associated with lineage divergence, whose consequences upon malaria transmission might be significant.

BackgroundUrban malaria is becoming a major health priority across Africa. A study was undertaken to assess the importance of urban pollution and agriculture practice on the distribution and susceptibility to insecticide of malaria vectors in the two main cities in Cameroon.MethodsAnopheline larval breeding sites were surveyed and water samples analysed monthly from October 2009 to December 2010. Parameters analysed included turbidity, pH, temperature, conductivity, sulfates, phosphates, nitrates, nitrites, ammonia, aluminium, alkalinity, iron, potassium, manganese, magnesium, magnesium hardness and total hardness. Characteristics of water bodies in urban areas were compared to rural areas and between urban sites. The level of susceptibility of Anopheles gambiae to 4% DDT, 0.75% permethrin, 0.05% deltamethrin, 0.1% bendiocarb and 5% malathion were compared between mosquitoes collected from polluted, non polluted and cultivated areas.ResultsA total of 1,546 breeding sites, 690 in Yaoundé and 856 in Douala, were sampled in the course of the study. Almost all measured parameters had a concentration of 2- to 100-fold higher in urban compare to rural breeding sites. No resistance to malathion was detected, but bendiocarb resistance was present in Yaounde. Very low mortality rates were observed following DDT or permethrin exposure, associated with high kdr frequencies. Mosquitoes collected in cultivated areas, exhibited the highest resistant levels. There was little difference in insecticide resistance or kdr allele frequency in mosquitoes collected from polluted versus non-polluted sites.ConclusionThe data confirm high selection pressure on mosquitoes originating from urban areas and suggest urban agriculture rather than pollution as the major factor driving resistance to insecticide.

Malaria still has a devastating impact on public health and welfare in Cameroon. Despite the increasing number of studies conducted on disease prevalence, transmission patterns or treatment, there are to date, not enough studies summarising findings from previous works in order to identify gaps in knowledge and areas of interest where further evidence is needed to drive malaria elimination efforts. The present study seeks to address these gaps by providing a review of studies conducted so far on malaria in Cameroon since the 1940s to date. Over 250 scientific publications were consulted for this purpose. Although there has been increased scale-up of vector control interventions which significantly reduced the morbidity and mortality to malaria across the country from a prevalence of 41% of the population reporting at least one malaria case episode in 2000 to a prevalence of 24% in 2017, the situation is not yet under control. There is a high variability in disease endemicity between epidemiological settings with prevalence of Plasmodium parasitaemia varying from 7 to 85% in children aged 6 months to 15 years after long-lasting insecticidal nets (LLINs) scale-up. Four species of Plasmodium have been recorded across the country: Plasmodium falciparum, P. malariae, P. ovale and P. vivax. Several primate-infecting Plasmodium spp. are also circulating in Cameroon. A decline of artemisinin-based combinations therapeutic efficacy from 97% in 2006 to 90% in 2016 have been reported. Several mutations in the P. falciparum chloroquine resistance (Pfcrt) and P. falciparum multidrug resistance 1 (Pfmdr1) genes conferring resistance to either 4-amino-quinoleine, mefloquine, halofanthrine and quinine have been documented. Mutations in the Pfdhfr and Pfdhps genes involved in sulfadoxine-pyrimethamine are also on the rise. No mutation associated with artemisinin resistance has been recorded. Sixteen anopheline species contribute to malaria parasite transmission with six recognized as major vectors: An. gambiae, An. coluzzii, An. arabiensis, An. funestus, An. nili and An. moucheti. Studies conducted so far, indicated rapid expansion of DDT, pyrethroid and carbamate resistance in An. gambiae, An. coluzzii, An. arabiensis and An. funestus threatening the performance of LLINs. This review highlights the complex situation of malaria in Cameroon and the need to urgently implement and reinforce integrated control strategies in different epidemiological settings, as part of the substantial efforts to consolidate gains and advance towards malaria elimination in the country.

BackgroundMalaria remains a major public health threat in Cameroon and disease prevention is facing strong challenges due to the rapid expansion of insecticide resistance in vector populations. The present review presents an overview of published data on insecticide resistance in the main malaria vectors in Cameroon to assist in the elaboration of future and sustainable resistance management strategies.MethodsA systematic search on mosquito susceptibility to insecticides and insecticide resistance in malaria vectors in Cameroon was conducted using online bibliographic databases including PubMed, Google and Google Scholar. From each peer-reviewed paper, information on the year of the study, mosquito species, susceptibility levels, location, insecticides, data source and resistance mechanisms were extracted and inserted in a Microsoft Excel datasheet. The data collected were then analysed for assessing insecticide resistance evolution.ResultsThirty-three scientific publications were selected for the analysis. The rapid evolution of insecticide resistance across the country was reported from 2000 onward. Insecticide resistance was highly prevalent in both An. gambiae (s.l.) and An. funestus. DDT, permethrin, deltamethrin and bendiocarb appeared as the most affected compounds by resistance. From 2000 to 2017 a steady increase in the prevalence of kdr allele frequency was noted in almost all sites in An. gambiae (s.l.), with the L1014F kdr allele being the most prevalent. Several detoxification genes (particularly P450 monooxygenase) were associated with DDT, pyrethroids and bendiocarb resistance. In An. funestus, resistance to DDT and pyrethroids was mainly attributed to the 119F-GSTe2 metabolic resistance marker and over-expression of P450 genes whereas the 296S-RDL mutation was detected in dieldrin-resistant An. funestus.ConclusionsThe review provides an update of insecticide resistance status in malaria vector populations in Cameroon and stresses the need for further actions to reinforce malaria control strategies in the coming years.

BackgroundDespite the recent progress in establishing the patterns of insecticide resistance in the major malaria vector Anopheles funestus, Central African populations of this species remain largely uncharacterised. To bridge this important gap and facilitate the implementation of suitable control strategies against this vector, we characterised the resistance patterns of An. funestus population from northern Cameroon.Methods and FindingsCollection of indoor-resting female mosquitoes in Gounougou (northern Cameroon) in 2012 and 2015 revealed a predominance of An. funestus during dry season. WHO bioassays performed using F1 An. funestus revealed that the population was multiple resistant to several insecticide classes including pyrethroids (permethrin, deltamethrin, lambda-cyhalothrin and etofenprox), carbamates (bendiocarb) and organochlorines (DDT and dieldrin). However, a full susceptibility was observed against the organophosphate malathion. Bioassays performed with 2015 collection revealed that resistance against pyrethroids and DDT is increasing. PBO synergist assays revealed a significant recovery of susceptibility for all pyrethroids but less for DDT. Analysis of the polymorphism of a portion of the voltage-gated sodium channel gene (VGSC) revealed the absence of the L1014F/S kdr mutation but identified 3 novel amino acid changes I877L, V881L and A1007S. However, no association was established between VGSC polymorphism and pyrethroid/DDT resistance. The DDT resistant 119F-GSTe2 allele (52%) and the dieldrin resistant 296S-RDL allele (45%) were detected in Gounougou. Temporal analysis between 2006, 2012 and 2015 collections revealed that the 119F-GSTe2 allele was relatively stable whereas a significant decrease is observed for 296S-RDL allele.ConclusionThis multiple resistance coupled with the temporal increased in resistance intensity highlights the need to take urgent measures to prolong the efficacy of current insecticide-based interventions against An. funestus in this African region.

BackgroundIn the city of Yaoundé in Cameroon malaria is predominately transmitted by the M and S molecular forms of Anopheles gambiae and both are resistant to the pyrethroid insecticides and DDT. Mutations in the target site of these insecticides, present at a high frequency in malaria vectors in this city, contribute to this resistance profile. To identify additional resistance mechanisms, the expression profile of multiple DDT-resistant field populations of M and S molecular forms was compared to laboratory-susceptible populations.Methodology/Principal findingsThe prevalence of DDT resistance was highest in the S form population originating from the cultivated site of Nkolondom (mortality after WHO bioassay = 4%). A high prevalence of DDT resistance was also found in two urban M form populations, Messa from a pristine unpolluted environment (DDT mortality = 54%), and Gare, where the breeding sites are heavily polluted with organic matter (DDT mortality = 38%). Microarray analysis showed that several transcripts coding for detoxification enzymes (P450s, GSTs and UDPGTs) and ABC transporters were upregulated in the three populations. Despite the presence of multiple detoxification genes over expressed in the DDT-resistant subset of these field populations, only three were commonly over expressed in resistant populations from all three environments. Two of these genes, CYP6M2 and GSTD1-6, encode enzymes that have been previously shown to metabolize DDT.Conclusion/SignificanceAnalogous to target site resistance, genes involved in metabolic resistance to DDT are also shared between the M and S forms of An gambiae. Alternative explanations for this occurrence are explored.

BackgroundLimitations in the ability of organisms to tolerate environmental stressors affect their fundamental ecological niche and constrain their distribution to specific habitats. Evolution of tolerance, therefore, can engender ecological niche dynamics. Forest populations of the afro-tropical malaria mosquito Anopheles gambiae have been shown to adapt to historically unsuitable larval habitats polluted with decaying organic matter that are found in densely populated urban agglomerates of Cameroon. This process has resulted in niche expansion from rural to urban environments that is associated with cryptic speciation and ecological divergence of two evolutionarily significant units within this taxon, the molecular forms M and S, among which reproductive isolation is significant but still incomplete. Habitat segregation between the two forms results in a mosaic distribution of clinally parapatric patches, with the M form predominating in the centre of urban agglomerates and the S form in the surrounding rural localities. We hypothesized that development of tolerance to nitrogenous pollutants derived from the decomposition of organic matter, among which ammonia is the most toxic to aquatic organisms, may affect this pattern of distribution and process of niche expansion by the M form.ResultsAcute toxicity bioassays indicated that populations of the two molecular forms occurring at the extremes of an urbanization gradient in Yaounde, the capital of Cameroon, differed in their response to ammonia. The regression lines best describing the dose-mortality profile differed in the scale of the explanatory variable (ammonia concentration log-transformed for the S form and linear for the M form), and in slope (steeper for the S form and shallower for the M form). These features reflected differences in the frequency distribution of individual tolerance thresholds in the two populations as assessed by probit analysis, with the M form exhibiting a greater mean and variance compared to the S form.ConclusionsIn agreement with expectations based on the pattern of habitat partitioning and exposure to ammonia in larval habitats in Yaounde, the M form showed greater tolerance to ammonia compared to the S form. This trait may be part of the physiological machinery allowing forest populations of the M form to colonize polluted larval habitats, which is at the heart of its niche expansion in densely populated human settlements in Cameroon.

Understanding how divergent selection generates adaptive phenotypic and population diversification provides a mechanistic explanation of speciation in recently separated species pairs. Towards this goal, we sought ecological gradients of divergence between the cryptic malaria vectors Anopheles coluzzii and An. gambiae and then looked for a physiological trait that may underlie such divergence. Using a large set of occurrence records and eco-geographic information, we built a distribution model to predict the predominance of the two species across their range of sympatry. Our model predicts two novel gradients along which the species segregate: distance from the coastline and altitude. Anopheles coluzzii showed a ‘bimodal’ distribution, predominating in xeric West African savannas and along the western coastal fringe of Africa. To test whether differences in salinity tolerance underlie this habitat segregation, we assessed the acute dose–mortality response to salinity of thirty-two larval populations from Central Africa. In agreement with its coastal predominance, Anopheles coluzzii was overall more tolerant than An. gambiae. Salinity tolerance of both species, however, converged in urban localities, presumably reflecting an adaptive response to osmotic stress from anthropogenic pollutants. When comparing degree of tolerance in conjunction with levels of syntopy, we found evidence of character displacement in this trait.