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.

Association mapping desiccation resistance within chromosomal inversions in the African malaria vector Anopheles gambiae.

Inversion polymorphisms are responsible for many ecologically important phenotypes and are often found under balancing selection. However, the same features that ensure their large role in local adaptation-especially reduced recombination between alternate arrangements-mean that uncovering the precise loci within inversions that control these phenotypes is unachievable using standard mapping approaches. Here, we take advantage of long-term balancing selection on a pair of inversions in the mosquito Anopheles gambiae to map desiccation tolerance via pool-GWAS. Two polymorphic inversions on chromosome 2 of this species (denoted 2La and 2Rb) are associated with arid and hot conditions in Africa and are maintained in spatially and temporally heterogeneous environments. After measuring thousands of wild-caught individuals for survival under desiccation stress, we used phenotypically extreme individuals homozygous for alternative arrangements at the 2La inversion to construct pools for whole-genome sequencing. Genomewide association mapping using these pools revealed dozens of significant SNPs within both 2La and 2Rb, many of which neighboured genes controlling ion channels or related functions. Our results point to the promise of similar approaches in systems with inversions maintained by balancing selection and provide a list of candidate genes underlying the specific phenotypes controlled by the two inversions studied here.

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.

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.

Adaptation to aridity in the malaria mosquito Anopheles gambiae: chromosomal inversion polymorphism and body size influence resistance to desiccation.

Chromosomal inversions are thought to confer a selective advantage in alternative habitats by protecting co-adapted alleles from recombination. The frequencies of two inversions (2La and 2Rb) of the afro-tropical malaria mosquito Anopheles gambiae change gradually along geographical clines, increasing in frequency with degree of aridity. Such clines can result from gene flow and local selection acting upon alternative karyotypes along the cline, suggesting that these inversions may be associated with tolerance to xeric conditions. Since water loss represents a major challenge in xeric habitats, it can be supposed that genes inside these inversions are involved in water homeostasis. To test this hypothesis, we compared the desiccation resistance of alternative karyotypes from a colonised 2Rb/2La polymorphic population of A. gambiae from Cameroon. The strain included only the molecular form S, one of the genetic units marking incipient speciation in this taxon. Day-old mosquitoes of both sexes were assayed individually for time to death in a dry environment and the karyotype of each was determined post-mortem using molecular diagnostic assays for each inversion. In agreement with expectations based on their eco-geographical distribution, we found that 2La homokaryotypes survived significantly longer (1.3 hours) than the other karyotypes. However, there was weak support for the effect of 2Rb on desiccation resistance. Larger mosquitoes survived longer than smaller ones. Median survival of females was greater than males, but the effect of sex on desiccation resistance was weakly supported, indicating that differential survival was correlated to differences between sexes in average size. We found weak evidence for a heterotic effect of 2La karyotype on size in females. These results support the notion that genes located inside the 2La inversion are involved in water balance, contributing towards local adaptation of A. gambiae to xeric habitats, beyond the adaptive value conferred by a larger body size.

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.