Field-evolved resistance to neonicotinoids in the mosquito, Anopheles gambiae, is associated with downregulation and mutations of nicotinic acetylcholine receptor subunits combined with cytochrome P450-mediated detoxification.

Neonicotinoid insecticides act selectively on their nicotinic receptor targets leading to variable sensitivity among arthropods. This study aimed to investigate the molecular mechanisms underlying contrasting susceptibility to neonicotinoids observed in wild populations of two mosquito sibling species. Bioassays and a synergism test revealed that the sister taxa, Anopheles gambiae and An. coluzzii, from Yaounde, Cameroon, rely on cytochrome P450s to detoxify neonicotinoids and develop resistance. However, contrary to An. coluzzii, An. gambiae populations are evolving stronger resistance to several active ingredients facilitated by mutations and reduced expression of nicotinic acetylcholine receptors. Six mutations were detected in coding sequences of the ß1 and α6 subunits, including two substitutions in one of the loops that modulate ligand binding and sensitivity. Allele frequencies were strongly correlated with a susceptibility gradient between An. coluzzii and An. gambiae suggesting that the mutations may play a key role in sensitivity. Messenger RNA expression levels of the ß1, α3, and α7 subunits decreased dramatically, on average by 23.27, 17.50, 15.80-fold, respectively, in wild An. gambiae populations compared to a susceptible insectary colony. By contrast, only the ß2 and α9-1 subunits were moderately downregulated (5.28 and 2.67-fold change, respectively) in field-collected An. coluzzii adults relative to susceptible colonized mosquitoes. Our findings provide critical information for the application and resistance management of neonicotinoids in malaria prevention.

Anopheles gambiae larvae's ability to grow and emerge in water containing lethal concentrations of clothianidin, acetamiprid, or imidacloprid is consistent with cross-resistance to neonicotinoids.

BACKGROUND: For decades, various agrochemicals have been successfully repurposed for mosquito control. However, preexisting resistance caused in larval and adult populations by unintentional pesticide exposure or other cross-resistance mechanisms poses a challenge to the efficacy of this strategy. A better understanding of larval adaptation to the lethal and sublethal effects of residual pesticides in aquatic habitats would provide vital information for assessing the efficacy of repurposed agrochemicals against mosquitoes. METHODS: We reared field-collected mosquito larvae in water containing a concentration of agrochemical causing 100% mortality in susceptible mosquitoes after 24 h (lethal concentration). Using this experimental setup, we tested the effect of lethal concentrations of a pyrrole (chlorfenapyr, 0.10 mg/l), a pyrethroid (deltamethrin, 1.5 mg/l), and three neonicotinoids including imidacloprid (0.075 mg/l), acetamiprid (0.15 mg/l), and clothianidin (0.035 mg/l) on mortality rates, growth, and survival in third-instar larvae of the two sibling species Anopheles gambiae and Anopheles coluzzii collected from Yaoundé, Cameroon. RESULTS: We found that An. gambiae and An. coluzzii larvae were susceptible to chlorfenapyr and were killed within 24 h by a nominal concentration of 0.10 mg/l. Consistent with strong resistance, deltamethrin induced low mortality in both species. Lethal concentrations of acetamiprid, imidacloprid, and clothianidin strongly inhibited survival, growth, and emergence in An. coluzzii larvae. By contrast, depending on the active ingredient and the population tested, 5-60% of immature stages of An. gambiae were able to grow and emerge in water containing a lethal concentration of neonicotinoids, suggesting cross-resistance to this class of insecticides. CONCLUSIONS: These findings corroborate susceptibility profiles observed in adults and suggest that unintentional pesticide exposure or other cross-resistance processes could contribute to the development of resistance to neonicotinoids in some Anopheles populations.

Adult mosquitoes of the sibling species Anopheles gambiae and Anopheles coluzzii exhibit contrasting patterns of susceptibility to four neonicotinoid insecticides along an urban-to-rural gradient in Yaoundé, Cameroon.

BACKGROUND: Neonicotinoids are potential alternatives for controlling pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of sub-Saharan Africa has yet to be investigated. The aim of the present study was to test the efficacy of four neonicotinoids against adult populations of the sibling species Anopheles gambiae and Anopheles coluzzii sampled along an urban-to-rural gradient. METHODS: The lethal toxicity of three active ingredients for adults of two susceptible Anopheles strains was assessed using concentration-response assays, and their discriminating concentrations were calculated. The discriminating concentrations were then used to test the susceptibility of An. gambiae and An. coluzzii mosquitoes collected from urban, suburban and rural areas of Yaoundé, Cameroon, to acetamiprid, imidacloprid, clothianidin and thiamethoxam. RESULTS: Lethal concentrations of neonicotinoids were relatively high suggesting that this class of insecticides has low toxicity against Anopheles mosquitoes. Reduced susceptibility to the four neonicotinoids tested was detected in An. gambiae populations collected from rural and suburban areas. By contrast, adults of An. coluzzii that occurred in urbanized settings were susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. The cytochrome inhibitor, piperonyl butoxide (PBO), significantly enhanced the activity of clothianidin and acetamiprid against An. gambiae mosquitoes. CONCLUSIONS: These findings corroborate susceptibility profiles observed in larvae and highlight a significant variation in tolerance to neonicotinoids between An. gambiae and An. coluzzii populations from Yaoundé. Further studies are needed to disentangle the role of exposure to agricultural pesticides and of cross-resistance mechanisms in the development of neonicotinoid resistance in some Anopheles species.

Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying.

BACKGROUND: Chronic exposure of mosquito larvae to pesticide residues and cross-resistance mechanisms are major drivers of tolerance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid prequalified for Indoor Residual Spraying (IRS). METHODS: Using standard bioassays, we tested if reduced susceptibility to clothianidin can affect the efficacy of SumiShield® 50WG, one of four new IRS formulations containing clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp sampled from urban, suburban and agricultural areas of Yaoundé, Cameroon. RESULTS: We found that in this geographic area, the level of susceptibility to the active ingredient predicted the efficacy of SumiShield 50WG. This formulation was very potent against populations that reached 100% mortality within 72 h of exposure to a discriminating concentration of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield 50WG in An. gambiae adults collected from a farm where the spraying of the two neonicotinoids acetamiprid and imidacloprid for crop protection is likely driving resistance to clothianidin. CONCLUSIONS: Despite the relatively small geographic extend of the study, the findings suggest that cross-resistance may impact the efficacy of some new IRS formulations and that alternative compounds could be prioritized in areas where neonicotinoid resistance is emerging.

Vegetable oil-based surfactants are adjuvants that enhance the efficacy of neonicotinoid insecticides and can bias susceptibility testing in adult mosquitoes.

BACKGROUND: The standard operating procedure for testing the susceptibility of adult mosquitoes to neonicotinoid or butenolide insecticides recommends using a vegetable oil ester (Mero) as a surfactant. However, there is growing evidence that this adjuvant contains surfactants that can enhance insecticide activity, mask resistance and bias the bioassay. METHODOLOGY/PRINCIPAL FINDINGS: Using standard bioassays, we tested the effects of commercial formulations of vegetable oil-based surfactants similar to Mero on the activity of a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). We found that three different brands of linseed oil soap used as cleaning products drastically enhanced neonicotinoid activity in Anopheles mosquitoes. At 1% (v/v), the surfactant reduced the median lethal concentration, LC50, of clothianidin more than 10-fold both in susceptible and in resistant populations of Anopheles gambiae. At 1% or 0.5% (v/v), linseed oil soap restored the susceptibility of adult mosquitoes fully to clothianidin, thiamethoxam and imidacloprid and partially to acetamiprid. By contrast, adding soap to the active ingredient did not significantly affect the level of resistance to permethrin or deltamethrin suggesting that vegetable oil-based surfactants specifically enhance the potency of some classes of insecticides. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that surfactants are not inert ingredients, and their use in susceptibility testing may jeopardize the ability to detect resistance. Further research is needed to evaluate the potential, the limitations and the challenges of using some surfactants as adjuvants to enhance the potency of some chemicals applied in mosquito control.

Vegetable oil surfactants are synergists that can bias neonicotinoid susceptibility testing in adult mosquitoes.

Background: The standard operating procedure for testing the susceptibility of adult mosquitoes to clothianidin, a neonicotinoid, recommends using a vegetable oil ester as surfactant. However, it has not yet been determined if the surfactant is an inert ingredient or if it can act as a synergist and bias the test. Methodology/Principal Findings: Using standard bioassays, we tested the synergistic effects of a vegetable oil surfactant on a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). Three different formulations of linseed oil soap used as surfactant were far more effective than the standard insecticide synergist piperonyl butoxide in enhancing neonicotinoid activity in Anopheles mosquitoes. At the concentration used in the standard operating procedure (1% v/v), vegetable oil surfactants lead to more than 10-fold reduction in lethal concentrations, LC 50 and LC 99 , of clothianidin in a multi-resistant field population and in a susceptible strain of Anopheles gambiae . At 1% or 0.5% (v/v), the surfactant restored susceptibility to clothianidin, thiamethoxam and imidacloprid and increased mortality to acetamiprid from 43 ± 5.63% to 89 ± 3.25% (P<0.05) in resistant mosquitoes. By contrast, linseed oil soap had no effect on the level of resistance to permethrin and deltamethrin suggesting that the synergism of vegetable oil surfactants may be specific to neoniconoids. Conclusions/Significance: Our findings indicate that vegetable oil surfactants are not inert ingredients in neonicotinoid formulations, and their synergistic effects undermine the ability of standard testing procedures to detect early stages of resistance.

Enhancing the efficacy of neonicotinoids against mosquitoes and overcoming resistance issues.

Background: Neonicotinoids are potential alternatives for targeting pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of Sub-Saharan Africa has yet to be investigated. Here we tested and compared the efficacy of four neonicotinoids alone or in combination with a synergist against two major vectors of Plasmodium . Results: Using standard bioassays, we first assessed the lethal toxicity of three active ingredients against adults of two susceptible Anopheles strains and we determined discriminating doses for monitoring susceptibility in wild populations. We then tested the susceptibility of 5532 Anopheles mosquitoes collected from urban and rural areas of Yaoundé, Cameroon, to discriminating doses of acetamiprid, imidacloprid, clothianidin and thiamethoxam. We found that in comparison with some public health insecticides, neonicotinoids have high lethal concentration, LC 99 , reflecting their low toxicity to Anopheles mosquitoes. In addition to this reduced toxicity, resistance to the four neonicotinoids tested was detected in An. gambiae populations collected from agricultural areas where larvae are intensively exposed to crop-protection neonicotinoids. However, adults of another major vector that occurred in urbanized settings, An. coluzzii , were fully susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. Importantly, the cytochrome inhibitor, piperonyl butoxide (PBO), was very effective in enhancing the activity of clothianidin and acetamiprid providing opportunities to create potent neonicotinoid formulations against Anopheles . Conclusion: These findings suggest that to successfully repurpose agricultural neonicotinoids for malaria vector control, it is essential to use formulations containing synergists such as PBO or surfactants to ensure optimal efficacy.

Chronic exposure of mosquito larvae to pesticide residues endangers a new generation of agrochemicals repurposed for malaria prevention.

Agrochemicals have been successfully repurposed to control mosquitoes worldwide, but pesticides used in agriculture challenge their effectiveness by contaminating surface waters and helping larval populations develop resistance. Thus, knowledge of the lethal and sublethal effects of residual pesticide exposure on mosquitoes is critical for selecting effective insecticides. Here we implemented a new experimental approach to predict the efficacy of agricultural pesticides newly repurposed for malaria vector control. We mimicked insecticide resistance selection as it occurs in contaminated aquatic habitats by rearing field-collected mosquito larvae in water containing a dose of insecticide capable of killing individuals from a susceptible strain within 24 h. We then simultaneously monitored short-term lethal toxicity within 24 h and sublethal effects for 7 days. We found that due to chronic exposure to agricultural pesticides, some mosquito populations are currently pre-adapt to resist neonicotinoids if those were used in vector control. Larvae collected from rural and agricultural areas where neonicotinoid formulations are intensively used for insect pest management were able to survive, grow, pupate and emerge in water containing a lethal dose of acetamiprid, imidacloprid or clothianidin. These results emphasize the importance of addressing exposure of larval populations to formulations applied in agriculture prior to using agrochemicals against malaria vectors.

Resistance to clothianidin reduces the efficacy of SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying, against Anopheles gambiae.

Chronic exposure of mosquito larvae to pesticide residues in agricultural areas is often associated with evolution of resistance to insecticides used for vector control. This presents a concern for the efficacy of clothianidin, an agricultural neonicotinoid qualified for Indoor Residual Spraying (IRS). Using standard bioassays, we tested if reduced susceptibility to clothianidin affects the efficacy of SumiShield® 50WG, one of the two newly approved formulations, which contains 50% clothianidin. We simultaneously monitored susceptibility to clothianidin and to SumiShield® 50WG, testing adults of Anopheles gambiae, An. coluzzii and Culex sp collected from urban, suburban and agricultural areas of Yaoundé. We found that the level of susceptibility to the active ingredient predicted the efficacy of SumiShield® 50WG. This formulation was very potent against populations that achieved 100% mortality within 72 h of exposure to a discriminating dose of clothianidin. By contrast, mortality leveled off at 75.4 ± 3.5% within 7 days of exposure to SumiShield® 50WG in An. gambiae adults collected from a farm where spraying of acetamiprid and imidacloprid is driving cross-resistance to clothianidin. These findings indicate that more potent formulations of clothianidin or different insecticides should be prioritized in areas where resistance is emerging.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

Integrated Mosquito Management: Is Precision Control a Luxury or Necessity?

The versatility of mosquito species that spread emerging arthropod-borne viruses such as Zika has highlighted the urgent need to re-evaluate mosquito-control standards. The prospect of using precise knowledge of the geographic distribution and vector status of local populations to guide targeted interventions has gained renewed attention, but the feasibility and utility of such an approach remain to be investigated. Using the example of mosquito management in the USA, we present ideas for designing, monitoring, and assessing precision vector control tailored to different environmental and epidemiological settings. We emphasize the technical adjustments that could be implemented in mosquito-control districts to enable targeted control while strengthening traditional management.

Human Interventions: Driving Forces of Mosquito Evolution.

One of the most common strategies for controlling mosquito-borne diseases relies on the use of chemical pesticides to repel or kill the mosquito vector. Pesticide exposure interferes with several key biological functions in the mosquito and triggers a variety of adaptive responses whose underlying mechanisms are only partially elucidated. The availability of reference genome sequences opens up the possibility of tracking signatures of evolutionary changes, including the most recent, across the genomes of many vector species. In this review, we highlight the recent genomic changes, which contribute to the fascinating adaptation of malaria vectors of the sub-Saharan African region to intensive insecticide-based interventions. We emphasize the operational significance of detailed genomic knowledge for current monitoring and decision-making.

Dissecting functional components of reproductive isolation among closely related sympatric species of the Anopheles gambiae complex.

Explaining how and why reproductive isolation evolves and determining which forms of reproductive isolation have the largest impact on the process of population divergence are major goals in the study of speciation. By studying recent adaptive radiations in incompletely isolated taxa, it is possible to identify barriers involved at early divergence before other confounding barriers emerge after speciation is complete. Sibling species of the Anopheles gambiae complex offer opportunities to provide insights into speciation mechanisms. Here, we studied patterns of reproductive isolation among three taxa, Anopheles coluzzii, An. gambiae s.s. and Anopheles arabiensis, to compare its strength at different spatial scales, to dissect the relative contribution of pre- versus postmating isolation, and to infer the involvement of ecological divergence on hybridization. Because F1 hybrids are viable, fertile and not uncommon, understanding the dynamics of hybridization in this trio of major malaria vectors has important implications for how adaptations arise and spread across the group, and in planning studies of the safety and efficacy of gene drive as a means of malaria control. We first performed a systematic review and meta-analysis of published surveys reporting on hybrid prevalence, showing strong reproductive isolation at a continental scale despite geographically restricted exceptions. Second, we exploited our own extensive field data sets collected at a regional scale in two contrasting environmental settings, to assess: (i) levels of premating isolation; (ii) spatio/temporal and frequency-dependent dynamics of hybridization, (iii) relationship between reproductive isolation and ecological divergence and (iv) hybrid viability penalty. Results are in accordance with ecological speciation theory predicting a positive association between the strength of reproductive isolation and degree of ecological divergence, and indicate that postmating isolation does contribute to reproductive isolation among these species. Specifically, only postmating isolation was positively associated with ecological divergence, whereas premating isolation was correlated with phylogenetic distance.

Genomic insights into adaptive divergence and speciation among malaria vectors of the Anopheles nili group.

Ongoing speciation in the most important African malaria vectors gives rise to cryptic populations, which differ remarkably in their behavior, ecology, and capacity to vector malaria parasites. Understanding the population structure and the drivers of genetic differentiation among mosquitoes is crucial for effective disease control because heterogeneity within vector species contributes to variability in malaria cases and allow fractions of populations to escape control efforts. To examine population structure and the potential impacts of recent large-scale control interventions, we have investigated the genomic patterns of differentiation in mosquitoes belonging to the Anopheles nili group-a large taxonomic group that diverged ~3 Myr ago. Using 4,343 single nucleotide polymorphisms (SNPs), we detected strong population structure characterized by high-FST values between multiple divergent populations adapted to different habitats within the Central African rainforest. Delineating the cryptic species within the Anopheles nili group is challenging due to incongruence between morphology, ribosomal DNA, and SNP markers consistent with incomplete lineage sorting and/or interspecific gene flow. A very high proportion of loci are fixed (FST = 1) within the genome of putative species, which suggests that ecological and/or reproductive barriers are maintained by strong selection on a substantial number of genes.

Chromosome arm-specific patterns of polymorphism associated with chromosomal inversions in the major African malaria vector, Anopheles funestus.

Chromosomal inversions facilitate local adaptation of beneficial mutations and modulate genetic polymorphism, but the extent of their effects within the genome is still insufficiently understood. The genome of Anopheles funestus, a malaria mosquito endemic to sub-Saharan Africa, contains an impressive number of paracentric polymorphic inversions, which are unevenly distributed among chromosomes and provide an excellent framework for investigating the genomic impacts of chromosomal rearrangements. Here, we present results of a fine-scale analysis of genetic variation within the genome of two weakly differentiated populations of Anopheles funestus inhabiting contrasting moisture conditions in Cameroon. Using population genomic analyses, we found that genetic divergence between the two populations is centred on regions of the genome corresponding to three inversions, which are characterized by high values of FST , absolute sequence divergence and fixed differences. Importantly, in contrast to the 2L chromosome arm, which is collinear, nucleotide diversity is significantly reduced along the entire length of three autosome arms bearing multiple overlapping chromosomal rearrangements. These findings support the idea that interactions between reduced recombination and natural selection within inversions contribute to sculpt nucleotide polymorphism across chromosomes in An. funestus.

Pollutants and Insecticides Drive Local Adaptation in African Malaria Mosquitoes.

The Anopheles gambiae complex contains a number of highly anthropophilic mosquito species that have acquired exceptional ability to thrive in complex human habitats. Thus, examining the evolutionary history of this Afrotropical mosquito may yield vital information on the selective processes that occurred during the adaptation to human-dominated environments. We performed reduced representation sequencing on 941 mosquitoes of the Anopheles gambiae complex collected across four ecogeographic zones in Cameroon. We find evidence for genetic and geographic subdivision within An. coluzzii and An. gambiae sensu stricto-the two most significant malaria vectors in the region. Importantly, in both species, rural and urban populations are genetically differentiated. Genome scans reveal pervasive signatures of selection centered on genes involved in xenobiotic resistance. Notably, a selective sweep containing detoxification enzymes is prominent in urban mosquitoes that exploit polluted breeding sites. Overall, our study suggests that recent anthropogenic environmental modifications and widespread use of insecticides are driving population differentiation and local adaptation in vectors with potentially significant consequences for malaria epidemiology.

Extensive genetic diversity among populations of the malaria mosquito Anopheles moucheti revealed by population genomics.

Malaria vectors are exposed to intense selective pressures due to large-scale intervention programs that are underway in most African countries. One of the current priorities is therefore to clearly assess the adaptive potential of Anopheline populations, which is critical to understand and anticipate the response mosquitoes can elicit against such adaptive challenges. The development of genomic resources that will empower robust examinations of evolutionary changes in all vectors including currently understudied species is an inevitable step toward this goal. Here we constructed double-digest Restriction Associated DNA (ddRAD) libraries and generated 6461 Single Nucleotide Polymorphisms (SNPs) that we used to explore the population structure and demographic history of wild-caught Anopheles moucheti from Cameroon. The genome-wide distribution of allelic frequencies among samples best fitted that of an old population at equilibrium, characterized by a weak genetic structure and extensive genetic diversity, presumably due to a large long term effective population size. Estimates of FST and Linkage Disequilibrium (LD) across SNPs reveal a very low genetic differentiation throughout the genome and the absence of segregating LD blocks among populations, suggesting an overall lack of local adaptation. Our study provides the first investigation of the genetic structure and diversity in An. moucheti at the genomic scale. We conclude that, despite a weak genetic structure, this species has the potential to challenge current vector control measures and other rapid anthropogenic and environmental changes thanks to its great genetic diversity.

Gene expression divergence between malaria vector sibling species Anopheles gambiae and An. coluzzii from rural and urban Yaoundé Cameroon.

Divergent selection based on aquatic larval ecology is a likely factor in the recent isolation of two broadly sympatric and morphologically identical African mosquito species, the malaria vectors Anopheles gambiae and An. coluzzii. Population-based genome scans have revealed numerous candidate regions of recent positive selection, but have provided few clues as to the genetic mechanisms underlying behavioural and physiological divergence between the two species, phenotypes which themselves remain obscure. To uncover possible genetic mechanisms, we compared global transcriptional profiles of natural and experimental populations using gene-based microarrays. Larvae were sampled as second and fourth instars from natural populations in and around the city of Yaoundé, capital of Cameroon, where the two species segregate along a gradient of urbanization. Functional enrichment analysis of differentially expressed genes revealed that An. coluzzii--the species that breeds in more stable, biotically complex and potentially polluted urban water bodies--overexpresses genes implicated in detoxification and immunity relative to An. gambiae, which breeds in more ephemeral and relatively depauperate pools and puddles in suburbs and rural areas. Moreover, our data suggest that such overexpression by An. coluzzii is not a transient result of induction by xenobiotics in the larval habitat, but an inherent and presumably adaptive response to repeatedly encountered environmental stressors. Finally, we find no significant overlap between the differentially expressed loci and previously identified genomic regions of recent positive selection, suggesting that transcriptome divergence is regulated by trans-acting factors rather than cis-acting elements.

Anthropogenic habitat disturbance and ecological divergence between incipient species of the malaria mosquito Anopheles gambiae.

BACKGROUND: Anthropogenic 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 FINDINGS: In 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/SIGNIFICANCE: Our 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.

Spatially explicit analyses of anopheline mosquitoes indoor resting density: implications for malaria control.

BACKGROUND: The question of sampling and spatial aggregation of malaria vectors is central to vector control efforts and estimates of transmission. Spatial patterns of anopheline populations are complex because mosquitoes' habitats and behaviors are strongly heterogeneous. Analyses of spatially referenced counts provide a powerful approach to delineate complex distribution patterns, and contributions of these methods in the study and control of malaria vectors must be carefully evaluated. METHODOLOGY/PRINCIPAL FINDINGS: We used correlograms, directional variograms, Local Indicators of Spatial Association (LISA) and the Spatial Analysis by Distance IndicEs (SADIE) to examine spatial patterns of Indoor Resting Densities (IRD) in two dominant malaria vectors sampled with a 5 × 5 km grid over a 2500 km(2) area in the forest domain of Cameroon. SADIE analyses revealed that the distribution of Anopheles gambiae was different from regular or random, whereas there was no evidence of spatial pattern in Anopheles funestus (Ia = 1.644, Pa<0.05 and Ia = 1.464, Pa>0.05, respectively). Correlograms and variograms showed significant spatial autocorrelations at small distance lags, and indicated the presence of large clusters of similar values of abundance in An. gambiae while An. funestus was characterized by smaller clusters. The examination of spatial patterns at a finer spatial scale with SADIE and LISA identified several patches of higher than average IRD (hot spots) and clusters of lower than average IRD (cold spots) for the two species. Significant changes occurred in the overall spatial pattern, spatial trends and clusters when IRDs were aggregated at the house level rather than the locality level. All spatial analyses unveiled scale-dependent patterns that could not be identified by traditional aggregation indices. CONCLUSIONS/SIGNIFICANCE: Our study illustrates the importance of spatial analyses in unraveling the complex spatial patterns of malaria vectors, and highlights the potential contributions of these methods in malaria control.