Intervention reducing malaria parasite load in vector mosquitoes: No impact on Plasmodium falciparum extrinsic incubation period and the survival of Anopheles gambiae.

In the fight against malaria, transmission blocking interventions (TBIs) such as transmission blocking vaccines or drugs, are promising approaches to complement conventional tools. They aim to prevent the infection of vectors and thereby reduce the subsequent exposure of a human population to infectious mosquitoes. The effectiveness of these approaches has been shown to depend on the initial intensity of infection in mosquitoes, often measured as the mean number of oocysts resulting from an infectious blood meal in absence of intervention. In mosquitoes exposed to a high intensity of infection, current TBI candidates are expected to be ineffective at completely blocking infection but will decrease parasite load and therefore, potentially also affect key parameters of vector transmission. The present study investigated the consequences of changes in oocyst intensity on subsequent parasite development and mosquito survival. To address this, we experimentally produced different intensities of infection for Anopheles gambiae females from Burkina Faso by diluting gametocytes from three natural Plasmodium falciparum local isolates and used a newly developed non-destructive method based on the exploitation of mosquito sugar feeding to track parasite and mosquito life history traits throughout sporogonic development. Our results indicate the extrinsic incubation period (EIP) of P. falciparum and mosquito survival did not vary with parasite density but differed significantly between parasite isolates with estimated EIP50 of 16 (95% CI: 15-18), 14 (95% CI: 12-16) and 12 (95% CI: 12-13) days and median longevity of 25 (95% CI: 22-29), 15 (95% CI: 13-15) and 18 (95% CI: 17-19) days for the three isolates respectively. Our results here do not identify unintended consequences of the decrease of parasite loads in mosquitoes on the parasite incubation period or on mosquito survival, two key parameters of vectorial capacity, and hence support the use of transmission blocking strategies to control malaria.

Mosquito ageing modulates the development, virulence and transmission potential of pathogens.

Host age variation is a striking source of heterogeneity that can shape the evolution and transmission dynamic of pathogens. Compared with vertebrate systems, our understanding of the impact of host age on invertebrate-pathogen interactions remains limited. We examined the influence of mosquito age on key life-history traits driving human malaria transmission. Females of Anopheles coluzzii, a major malaria vector, belonging to three age classes (4-, 8- and 12-day-old), were experimentally infected with Plasmodium falciparum field isolates. Our findings revealed reduced competence in 12-day-old mosquitoes, characterized by lower oocyst/sporozoite rates and intensities compared with younger mosquitoes. Despite shorter median longevities in older age classes, infected 12-day-old mosquitoes exhibited improved survival, suggesting that the infection might act as a fountain of youth for older mosquitoes specifically. The timing of sporozoite appearance in the salivary glands remained consistent across mosquito age classes, with an extrinsic incubation period of approximately 13 days. Integrating these results into an epidemiological model revealed a lower vectorial capacity for older mosquitoes compared with younger ones, albeit still substantial owing to extended longevity in the presence of infection. Considering age heterogeneity provides valuable insights for ecological and epidemiological studies, informing targeted control strategies to mitigate pathogen transmission.

A simple, field-applicable method to increase the infectivity of wild isolates of Plasmodium falciparum to mosquito vectors.

BACKGROUND: The direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes. METHODS: Anopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence. RESULTS: Substituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control. CONCLUSIONS: Concentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions.

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies.

Plasmodium falciparum malaria continues to evade control efforts, utilizing highly specialized sexual-stages to transmit infection between the human host and mosquito vector. In a vaccination model, antibodies directed to sexual-stage antigens, when ingested in the mosquito blood meal, can inhibit parasite growth in the midgut and consequently arrest transmission. Despite multiple datasets for the Plasmodium sexual-stage transcriptome and proteome, there have been no rational screens to identify candidate antigens for transmission-blocking vaccine (TBV) development. This study characterizes 12 proteins from across the P. falciparum sexual-stages as possible TBV targets. Recombinant proteins are heterologously expressed as full-length ectodomains in a mammalian HEK293 cell system. The proteins recapitulate native parasite epitopes as assessed by indirect fluorescence assay and a proportion exhibits immunoreactivity when tested against sera from individuals living in malaria-endemic Burkina Faso and Mali. Purified IgG generated to the mosquito-stage parasite antigen enolase demonstrates moderate inhibition of parasite development in the mosquito midgut by the ex vivo standard membrane feeding assay. The findings support the use of rational screens and comparative functional assessments in identifying proteins of the P. falciparum transmission pathway and establishing a robust pre-clinical TBV pipeline.

Effect of seasonal malaria chemoprevention plus azithromycin on Plasmodium falciparum transmission: gametocyte infectivity and mosquito fitness.

BACKGROUND: Seasonal malaria chemoprevention (SMC) consists of administration of sulfadoxine-pyrimethamine (SP) + amodiaquine (AQ) at monthly intervals to children during the malaria transmission period. Whether the addition of azithromycin (AZ) to SMC could potentiate the benefit of the intervention was tested through a double-blind, randomized, placebo-controlled trial. The effect of SMC and the addition of AZ, on malaria transmission and on the life history traits of Anopheles gambiae mosquitoes have been investigated. METHODS: The study included 438 children randomly selected from among participants in the SMC + AZ trial and 198 children from the same area who did not receive chemoprevention. For each participant in the SMC + AZ trial, blood was collected 14 to 21 days post treatment, examined for the presence of malaria sexual and asexual stages and provided as a blood meal to An. gambiae females using a direct membrane-feeding assay. RESULTS: The SMC treatment, with or without AZ, significantly reduced the prevalence of asexual Plasmodium falciparum (LRT X22 = 69, P < 0.0001) and the gametocyte prevalence (LRT X22 = 54, P < 0.0001). In addition, the proportion of infectious feeds (LRT X22 = 61, P < 0.0001) and the prevalence of oocysts among exposed mosquitoes (LRT X22 = 22.8, P < 0.001) was reduced when mosquitoes were fed on blood from treated children compared to untreated controls. The addition of AZ to SPAQ was associated with an increased proportion of infectious feeds (LRT X21 = 5.2, P = 0.02), suggesting a significant effect of AZ on gametocyte infectivity. There was a slight negative effect of SPAQ and SPAQ + AZ on mosquito survival compared to mosquitoes fed with blood from control children (LRTX22 = 330, P < 0.0001). CONCLUSION: This study demonstrates that SMC may contribute to a reduction in human to mosquito transmission of P. falciparum, and the reduced mosquito longevity observed for females fed on treated blood may increase the benefit of this intervention in control of malaria. The addition of AZ to SPAQ in SMC appeared to enhance the infectivity of gametocytes providing further evidence that this combination is not an appropriate intervention.

Mosquito Attractants.

Vector control and personal protection against anthropophilic mosquitoes mainly rely on the use of insecticides and repellents. The search for mosquito-attractive semiochemicals has been the subject of intense studies for decades, and new compounds or odor blends are regularly proposed as lures for odor-baited traps. We present a comprehensive and up-to-date review of all the studies that have evaluated the attractiveness of volatiles to mosquitoes, including individual chemical compounds, synthetic blends of compounds, or natural host or plant odors. A total of 388 studies were analysed, and our survey highlights the existence of 105 attractants (77 volatile compounds, 17 organism odors, and 11 synthetic blends) that have been proved effective in attracting one or several mosquito species. The exhaustive list of these attractants is presented in various tables, while the most common mosquito attractants - for which effective attractiveness has been demonstrated in numerous studies - are discussed throughout the text. The increasing knowledge on compounds attractive to mosquitoes may now serve as the basis for complementary vector control strategies, such as those involving lure-and-kill traps, or the development of mass trapping. This review also points out the necessity of further improving the search for new volatile attractants, such as new compound blends in specific ratios, considering that mosquito attraction to odors may vary over the life of the mosquito or among species. Finally, the use of mosquito attractants will undoubtedly have an increasingly important role to play in future integrated vector management programs.

Plant-Mediated Effects on Mosquito Capacity to Transmit Human Malaria.

The ecological context in which mosquitoes and malaria parasites interact has received little attention, compared to the genetic and molecular aspects of malaria transmission. Plant nectar and fruits are important for the nutritional ecology of malaria vectors, but how the natural diversity of plant-derived sugar sources affects mosquito competence for malaria parasites is unclear. To test this, we infected Anopheles coluzzi, an important African malaria vector, with sympatric field isolates of Plasmodium falciparum, using direct membrane feeding assays. Through a series of experiments, we then examined the effects of sugar meals from Thevetia neriifolia and Barleria lupilina cuttings that included flowers, and fruit from Lannea microcarpa and Mangifera indica on parasite and mosquito traits that are key for determining the intensity of malaria transmission. We found that the source of plant sugar meal differentially affected infection prevalence and intensity, the development duration of the parasites, as well as the survival and fecundity of the vector. These effects are likely the result of complex interactions between toxic secondary metabolites and the nutritional quality of the plant sugar source, as well as of host resource availability and parasite growth. Using an epidemiological model, we show that plant sugar source can be a significant driver of malaria transmission dynamics, with some plant species exhibiting either transmission-reducing or -enhancing activities.

Non-genetic determinants of mosquito competence for malaria parasites.

Understanding how mosquito vectors and malaria parasites interact is of fundamental interest, and it also offers novel perspectives for disease control. Both the genetic and environmental contexts are known to affect the ability of mosquitoes to support malaria development and transmission, i.e., vector competence. Although the role of environment has long been recognized, much work has focused on host and parasite genetic effects. However, the last few years have seen a surge of studies revealing a great diversity of ways in which non-genetic factors can interfere with mosquito-Plasmodium interactions. Here, we review the current evidence for such environmentally mediated effects, including ambient temperature, mosquito diet, microbial gut flora, and infection history, and we identify additional factors previously overlooked in mosquito-Plasmodium interactions. We also discuss epidemiological implications, and the evolutionary consequences for vector immunity and parasite transmission strategies. Finally, we propose directions for further research and argue that an improved knowledge of non-genetic influences on mosquito-Plasmodium interactions could aid in implementing conventional malaria control measures and contribute to the design of novel strategies.

Experimental study of the relationship between Plasmodium gametocyte density and infection success in mosquitoes; implications for the evaluation of malaria transmission-reducing interventions.

The evaluation of transmission reducing interventions (TRI) to control malaria widely uses membrane feeding assays. In such assays, the intensity of Plasmodium infection in the vector might affect the measured efficacy of the candidates to block transmission. Gametocyte density in the host blood is a determinant of the infection success in the mosquito, however, uncertain estimates of parasite densities and intrinsic characteristics of the infected blood can induce variability. To reduce this variation, a feasible method is to dilute infectious blood samples. We describe the effect of diluting samples of Plasmodium-containing blood samples to allow accurate relative measures of gametocyte densities and their impact on mosquito infectivity and TRI efficacy. Natural Plasmodium falciparum samples were diluted to generate a wide range of parasite densities, and fed to Anopheles coluzzii mosquitoes. This was compared with parallel dilutions conducted on Plasmodium berghei infections. We examined how blood dilution influences the observed blocking activity of anti-Pbs28 monoclonal antibody using the P. berghei/Anopheles stephensi system. In the natural species combination P. falciparum/An. coluzzii, blood dilution using heat-inactivated, infected blood as diluents, revealed positive near linear relationships, between gametocyte densities and oocyst loads in the range tested. A similar relationship was observed in the P. berghei/An. stephensi system when using a similar dilution method. In contrast, diluting infected mice blood with fresh uninfected blood dramatically increases the infectiousness. This suggests that highly infected mice blood contains inhibitory factors or reduced blood moieties, which impede infection and may in turn, lead to misinterpretation when comparing individual TRI evaluation assays. In the lab system, the transmission blocking activity of an antibody specific for Pbs28 was confirmed to be density-dependent. This highlights the need to carefully interpret evaluations of TRI candidates, regarding gametocyte densities in the P. berghei/An. stephensi system.

Interplay between Plasmodium infection and resistance to insecticides in vector mosquitoes.

Despite its epidemiological importance, the impact of insecticide resistance on vector-parasite interactions and malaria transmission is poorly understood. Here, we explored the impact of Plasmodium infection on the level of insecticide resistance to dichlorodiphenyltrichloroethane (DDT) in field-caught Anopheles gambiae sensu stricto homozygous for the kdr mutation. Results showed that kdr homozygous mosquitoes that fed on infectious blood were more susceptible to DDT than mosquitoes that fed on noninfectious blood during both ookinete development (day 1 after the blood meal) and oocyst maturation (day 7 after the blood meal) but not during sporozoite invasion of the salivary glands. Plasmodium falciparum infection seemed to impose a fitness cost on mosquitoes by reducing the ability of kdr homozygous A. gambiae sensu stricto to survive exposure to DDT. These results suggest an interaction between Plasmodium infection and the insecticide susceptibility of mosquitoes carrying insecticide-resistant alleles. We discuss this finding in relation to vector control efficacy.

Insecticide exposure impacts vector-parasite interactions in insecticide-resistant malaria vectors.

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles-Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.

A heavy legacy: offspring of malaria-infected mosquitoes show reduced disease resistance.

BACKGROUND: Trans-generational effects of immune stimulation may have either adaptive (trans-generational immune priming) or non-adaptive (fitness costs) effects on offspring ability to fight pathogens. METHODS: Anopheles coluzzii and its natural malaria parasite Plasmodium falciparum were used to test how maternal parasite infection affected offspring resistance to the same parasite species. RESULTS: Daughters of exposed mothers had similar qualitative resistance, as measured by their ability to prevent infection, relative to those of control mothers. However, maternal disease exposure altered offspring quantitative resistance, measured as the ability to limit parasite development, with mosquitoes of infected mothers suffering slightly increased parasite intensity compared to controls. In addition, quantitative resistance was minimal in offspring of highly infected mothers, and in offspring issued from eggs produced during the early infection phase. CONCLUSIONS: Plasmodium falciparum infection in An. coluzzii can have trans-generational costs, lowering quantitative resistance in offspring of infected mothers. Malaria-exposed mosquitoes might heavily invest in immune defences and thereby produce lower quality offspring that are poorly resistant.

Non-contact detection of pyrethroids widely used in vector control by Anopheles mosquitoes.

Pyrethroids are the most widely used insecticides to control vector borne diseases including malaria. Physiological resistance mechanisms to these insecticides have been well described, whereas those for behavioral resistance remain overlooked. Field data suggest the presence of spatial sensory detection by Anopheles mosquitoes of the pyrethroid molecules used in insecticide-based control tools, such as long-lasting insecticide nets or insecticide residual spraying. This opens the way to the emergence of a wide range of behavioral adaptations among malaria vectors. However, the spatial sensory detection of these molecules is controversial and needs to be demonstrated. The goal of this study was to behaviorally characterize the non-contact detection of three of the most common pyrethroids used for malaria vector control: permethrin, deltamethrin an ⍺-cypermethrin. To reach this goal, we recorded the behavior (takeoff response) of Anopheles gambiae pyrethroid-sensitive and resistant laboratory strains, as well as field collected mosquitoes from the Gambiae Complex, when exposed to the headspace of bottles containing different doses of the insecticides at 25 and 35°C, in order to represent a range of laboratory and field temperatures. We found the proportion of laboratory susceptible and resistant female mosquitoes that took off was, in all treatments, dose and the temperature dependent. Sensitive mosquitoes were significantly more prone to take off only in the presence of ⍺-cypermethrin, whereas sensitive and resistant mosquitoes showed similar responses to permethrin and deltamethrin. Field-collected mosquitoes of the Gambiae Complex were also responsive to permethrin, independently of the species identity (An. gambiae, An. coluzzii and An. arabiensis) or their genotypes for the kdr mutation, known to confer resistance to pyrethroids. The observed ability of Anopheles spp. mosquitoes to detect insecticides without contact could favor the evolution of behavioral modifications that may allow them to avoid or reduce the adverse effect of insecticides and thus, the development of behavioral resistance.

The paradox of plant preference: The malaria vectors Anopheles gambiae and Anopheles coluzzii select suboptimal food sources for their survival and reproduction.

Anopheles gambiae and Anopheles coluzzii mosquitoes, two major malaria vectors in sub-Saharan Africa, exhibit selectivity among plant species as potential food sources. However, it remains unclear if their preference aligns with optimal nutrient intake and survival. Following an extensive screening of the effects of 31 plant species on An. coluzzii in Burkina Faso, we selected three species for their contrasting effects on mosquito survival, namely Ixora coccinea, Caesalpinia pulcherrima, and Combretum indicum. We assessed the sugar content of these plants and their impact on mosquito fructose positivity, survival, and insemination rate, using Anopheles coluzzii and Anopheles gambiae, with glucose 5% and water as controls. Plants displayed varying sugar content and differentially affected the survival, sugar intake, and insemination rate of mosquitoes. All three plants were more attractive to mosquitoes than controls, with An. gambiae being more responsive than An. coluzzii. Notably, C. indicum was the most attractive but had the lowest sugar content and offered the lowest survival, insemination rate, and fructose positivity. Our findings unveil a performance-preference mismatch in An. coluzzii and An. gambiae regarding plant food sources. Several possible reasons for this negative correlation between performance and preference are discussed.

Anti-Pfs25 human plasma reduces transmission of Plasmodium falciparum isolates that have diverse genetic backgrounds.

Pfs25 is a leading candidate for a malaria transmission-blocking vaccine whose potential has been demonstrated in a phase 1 trial with recombinant Pfs25 formulated with Montanide ISA51. Because of limited sequence polymorphism, the anti-Pfs25 antibodies induced by this vaccine are likely to have transmission-blocking or -reducing activity against most, if not all, field isolates. To test this hypothesis, we evaluated transmission-blocking activities by membrane feeding assay of anti-Pfs25 plasma from the Pfs25/ISA51 phase 1 trial against Plasmodium falciparum parasites from patients in two different geographical regions of the world, Thailand and Burkina Faso. In parallel, parasite isolates from these patients were sequenced for the Pfs25 gene and genotyped for seven microsatellites. The results indicate that despite different genetic backgrounds among parasite isolates, the Pfs25 sequences are highly conserved, with a single nonsynonymous nucleotide polymorphism detected in 1 of 41 patients in Thailand and Burkina Faso. The anti-Pfs25 immune plasma had significantly higher transmission-reducing activity against parasite isolates from the two geographical regions than the nonimmune controls (P < 0.0001).

New methods for field collection of human skin volatiles and perspectives for their application in the chemical ecology of human-pathogen-vector interactions.

Odours emitted by human skin are of great interest to biologists in many fields, with practical applications in forensics, health diagnostic tools and the ecology of blood-sucking insect vectors of human disease. Convenient methods are required for sampling human skin volatiles under field conditions. We experimentally compared four modern methods for sampling skin odours: solvent extraction, headspace solid-phase micro-extraction (SPME), and two new techniques not previously used for the study of mammal volatiles, contact SPME and dynamic headspace with a chromatoprobe design. These methods were tested and compared both on European subjects under laboratory conditions and on young African subjects under field conditions. All four methods permitted effective trapping of skin odours, including the major known human skin volatile compounds. In both laboratory and field experiments, contact SPME, in which the time of collection was restricted to 3 min, provided results very similar to those obtained with classical headspace SPME, a method that requires 45 min of collection. Chromatoprobe sampling also proved to be very sensitive, rapid and convenient for the collection of human-produced volatiles in natural settings. Both contact SPME and chromatoprobe design may considerably facilitate the study of human skin volatiles under field conditions, opening new possibilities for examining the olfactory cues mediating the host-seeking behaviour of mosquito vectors implicated in the transmission of major diseases.

Studying fitness cost of Plasmodium falciparum infection in malaria vectors: validation of an appropriate negative control.

BACKGROUND: The question whether Plasmodium falciparum infection affects the fitness of mosquito vectors remains open. A hurdle for resolving this question is the lack of appropriate control, non-infected mosquitoes that can be compared to the infected ones. It was shown recently that heating P. falciparum gametocyte-infected blood before feeding by malaria vectors inhibits the infection. Therefore, the same source of gametocyte-infected blood could be divided in two parts, one heated, serving as the control, the other unheated, allowing the comparison of infected and uninfected mosquitoes which fed on exactly the same blood otherwise. However, before using this method for characterizing the cost of infection to mosquitoes, it is necessary to establish whether feeding on previously heated blood affects the survival and fecundity of mosquito females. METHODS: Anopheles gambiae M molecular form females were exposed to heated versus non-heated, parasite-free human blood to mimic blood meal on non-infectious versus infectious gametocyte-containing blood. Life history traits of mosquito females fed on blood that was heat-treated or not were then compared. RESULTS: The results reveal that heat treatment of the blood did not affect the survival and fecundity of mosquito females. Consistently, blood heat treatment did not affect the quantity of blood ingested. CONCLUSIONS: The study indicates that heat inactivation of gametocyte-infected blood will only inhibit mosquito infection and that this method is suitable for quantifying the fitness cost incurred by mosquitoes upon infection by P. falciparum.

Human skin volatiles: a review.

Odors emitted by human skin are of great interest to biologists in many fields; applications range from forensic studies to diagnostic tools, the design of perfumes and deodorants, and the ecology of blood-sucking insect vectors of human disease. Numerous studies have investigated the chemical composition of skin odors, and various sampling methods have been used for this purpose. The literature shows that the chemical profile of skin volatiles varies greatly among studies, and the use of different sampling procedures is probably responsible for some of these variations. To our knowledge, this is the first review focused on human skin volatile compounds. We detail the different sampling techniques, each with its own set of advantages and disadvantages, which have been used for the collection of skin odors from different parts of the human body. We present the main skin volatile compounds found in these studies, with particular emphasis on the most frequently studied body regions, axillae, hands, and feet. We propose future directions for promising experimental studies on odors from human skin, particularly in relation to the chemical ecology of blood-sucking insects.

Chromosomal inversions, natural selection and adaptation in the malaria vector Anopheles funestus.

Chromosomal polymorphisms, such as inversions, are presumably involved in the rapid adaptation of populations to local environmental conditions. Reduced recombination between alternative arrangements in heterozygotes may protect sets of locally adapted genes, promoting ecological divergence and potentially leading to reproductive isolation and speciation. Through a comparative analysis of chromosomal inversions and microsatellite marker polymorphisms, we hereby present biological evidence that strengthens this view in the mosquito Anopheles funestus s.s, one of the most important and widespread malaria vectors in Africa. Specimens were collected across a wide range of geographical, ecological, and climatic conditions in Cameroon. We observed a sharp contrast between population structure measured at neutral microsatellite markers and at chromosomal inversions. Microsatellite data detected only a weak signal for population structuring among geographical zones (F(ST) < 0.013, P < 0.01). By contrast, strong differentiation among ecological zones was revealed by chromosomal inversions (F(ST) > 0.190, P < 0.01). Using standardized estimates of F(ST), we show that inversions behave at odds with neutral expectations strongly suggesting a role of environmental selection in shaping their distribution. We further demonstrate through canonical correspondence analysis that heterogeneity in eco-geographical variables measured at specimen sampling sites explained 89% of chromosomal variance in A. funestus. These results are in agreement with a role of chromosomal inversions in ecotypic adaptation in this species. We argue that this widespread mosquito represents an interesting model system for the study of chromosomal speciation mechanisms and should provide ample opportunity for comparative studies on the evolution of reproductive isolation and speciation in major human malaria vectors.