Mosquito Anopheles Gambiae Sensu Stricto Research Articles

Mosquito host preferences affect their response to synthetic and natural odour blends.

BackgroundThe anthropophilic malaria mosquito Anopheles gambiae sensu stricto (hereafter termed Anopheles gambiae) primarily takes blood meals from humans, whereas its close sibling Anopheles arabiensis is more opportunistic. Previous studies have identified several compounds that play a critical role in the odour-mediated behaviour of An. gambiae. This study determined the effect of natural and synthetic odour blends on mosquitoes with different host preferences to better understand the host-seeking behaviour of mosquitoes and the potential of synthetic odour blends for standardized monitoring.MethodsOdour blends were initially tested for their attractiveness to An. gambiae and An. arabiensis in a semi-field system with MM-X traps baited with natural and synthetic odours. Natural host odours were collected from humans, cows and chickens. The synthetic odour blends consisted of three or five previously identified compounds released with carbon dioxide. These studies were continued under natural conditions where odour blends were tested outdoors to determine their effect on species with different host preferences.ResultsIn the semi-field experiments, human odour attracted significantly higher numbers of both mosquito species. However, An. arabiensis was also attracted to cow and chicken odours, which confirms its opportunistic behaviour. A five-component synthetic blend was highly attractive to both mosquito species. In the field, the synthetic odour blend caught significantly more An. funestus than traps baited with human odour, while no difference was found for An. arabiensis. Catches of An. arabiensis and Culex spp. contained large numbers of blood-fed mosquitoes, mostly from cows, which indicates that these mosquitoes had fed outdoors.ConclusionsDifferent odour baits elicit varying responses among mosquito species. Synthetic odour blends are highly effective for trapping mosquitoes; however, not all mosquitoes respond equally to the same odour blend. Combining fermenting molasses with synthetic blends in a trap represents the most effective tool to catch blood-fed mosquitoes outside houses, which is essential for understanding outdoor malaria transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0635-1) contains supplementary material, which is available to authorized users.

Assessing the efficacy of candidate mosquito repellents against the background of an attractive source that mimics a human host.

Mosquito repellents are used around the globe to protect against nuisance biting and disease-transmitting mosquitoes. Recently, there has been renewed interest in the development of repellents as tools to control the transmission of mosquito-borne diseases. We present a new bioassay for the accurate assessment of candidate repellent compounds, using a synthetic odour that mimics the odour blend released by human skin. Using DEET (N,N-diethyl-meta-toluamide) and PMD (p-menthane-3,8-diol) as reference compounds, nine candidate repellents were tested, of which five showed significant repellency to the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae). These included: 2-nonanone; 6-methyl-5-hepten-2-one; linalool; δ-decalactone, and δ-undecalactone. The lactones were also tested on the yellow fever mosquito Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae), against which they showed similar degrees of repellency. We conclude that the lactones are highly promising repellents, particularly because these compounds are pleasant-smelling, natural products that are also present in human food sources.

Advances in methods for colour marking of mosquitoes

BackgroundDifferent techniques are available for colour marking insects and each technique may be suitable for different insect species. Mosquitoes can be marked to determine population size, distribution and flight distance or distinguish closely related species. In this study, two methods of colour marking mosquitoes were described in detail and the impact of both methods on the survival and host-seeking behaviour of the malaria mosquito Anopheles gambiae sensu stricto was investigated.MethodsMosquitoes were marked in groups with fluorescent powder or fluorescent dye. The powder was applied by creating a cloud of powder in a paper cup and the dye was applied with an airbrush. The effect of marking on the survival of mosquitoes of different age groups was tested under controlled conditions. The effect of marking on the host seeking response of the mosquitoes was tested in an olfactometer with human and cow odour as baits.ResultsNo effect of either of the marking methods was found on the survival of mosquitoes that were treated 1 or 3 days after emergence, however, the survival of mosquitoes treated 5 or 9 days after emergence was significantly reduced. The host-seeking response of mosquitoes to human or cow odour was tested in a dual-port olfactometer and was not found to be affected by treatment with fluorescent powder or dye.ConclusionsBoth methods are suitable for colour marking large groups of mosquitoes. Marking with fluorescent powder, however, is preferred because the method is simpler, visible without a UV light and no specific materials are required.

An age–size reaction norm yields insight into environmental interactions affecting life‐history traits: a factorial study of larval development in the malaria mosquito Anopheles gambiae sensu stricto

Environmental factors frequently act nonindependently to determine growth and development of insects. Because age and size at maturity strongly influence population dynamics, interaction effects among environmental variables complicate the task of predicting dynamics of insect populations under novel conditions. We reared larvae of the African malaria mosquito Anopheles gambiae sensu stricto (s.s.) under three factors relevant to changes in climate and land use: food level, water depth, and temperature. Each factor was held at two levels in a fully crossed design, for eight experimental treatments. Larval survival, larval development time, and adult size (wing length) were measured to indicate the importance of interaction effects upon population-level processes. For age and size at emergence, but not survival, significant interaction effects were detected for all three factors, in addition to sex. Some of these interaction effects can be understood as consequences of how the different factors influence energy usage in the context of a nonindependent relationship between age and size. Experimentally assessing interaction effects for all potential future sets of conditions is intractable. However, considering how different factors affect energy usage within the context of an insect's evolved developmental program can provide insight into the causes of complex environmental effects on populations.

A 3D Analysis of Flight Behavior of Anopheles gambiae sensu stricto Malaria Mosquitoes in Response to Human Odor and Heat

Female mosquitoes use odor and heat as cues to navigate to a suitable landing site on their blood host. The way these cues affect flight behavior and modulate anemotactic responses, however, is poorly understood. We studied in-flight behavioral responses of females of the nocturnal malaria mosquito Anopheles gambiae sensu stricto to human odor and heat. Flight-path characteristics in a wind tunnel (flow 20 cm/s) were quantified in three dimensions. With wind as the only stimulus (control), short and close to straight upwind flights were recorded. With heat alone, flights were similarly short and direct. The presence of human odor, in contrast, caused prolonged and highly convoluted flight patterns. The combination of odor+heat resulted in longer flights with more landings on the source than to either cue alone. Flight speed was greatest (mean groundspeed 27.2 cm/s) for odor+heat. Odor alone resulted in decreased flight speed when mosquitoes arrived within 30 cm of the source whereas mosquitoes exposed to odor+heat maintained a high flight speed while flying in the odor plume, until they arrived within 15 cm of the source. Human odor evoked an increase in crosswind flights with an additive effect of heat at close range (<15 cm) to the source. This was found for both horizontal and vertical flight components. However, mosquitoes nevertheless made upwind progress when flying in the odor+heat generated plume, suggesting that mosquitoes scan their environment intensively while they progress upwind towards their host. These observations may help to improve the efficacy of trapping systems for malaria mosquitoes by (1) optimizing the site of odor release relative to trap entry and (2) adding a heat source which enhances a landing response.

Breakdown in the process of incipient speciation in Anopheles gambiae.

Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea-Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) microarray enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form-specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing.

Behavioural responses of Anopheles gambiae sensu stricto M and S molecular form larvae to an aquatic predator in Burkina Faso

BackgroundPredation of aquatic immature stages has been identified as a major evolutionary force driving habitat segregation and niche partitioning in the malaria mosquito Anopheles gambiae sensu stricto in the humid savannahs of Burkina Faso, West Africa. Here, we explored behavioural responses to the presence of a predator in wild populations of the M and S molecular forms of An. gambiae that typically breed in permanent (e.g., rice field paddies) and temporary (e.g., road ruts) water collections.MethodsLarvae used in these experiments were obtained from eggs laid by wild female An. gambiae collected from two localities in south-western Burkina Faso during the 2008 rainy season. Single larvae were observed in an experimental arena, and behavioural traits were recorded and quantified a) in the absence of a predator and b) in the presence of a widespread mosquito predator, the backswimmer Anisops jaczewskii. Differences in the proportion of time allocated to each behaviour were assessed using Principal Component Analysis and Multivariate Analysis of Variance.ResultsThe behaviour of M and S form larvae was found to differ significantly; although both forms mainly foraged at the water surface, spending 60-90% of their time filtering water at the surface or along the wall of the container, M form larvae spent on average significantly more time browsing at the bottom of the container than S form larvae (4.5 vs. 1.3% of their overall time, respectively; P < 0.05). In the presence of a predator, larvae of both forms modified their behaviour, spending significantly more time resting along the container wall (P < 0.001). This change in behaviour was at least twice as great in the M form (from 38.6 to 66.6% of the time at the wall in the absence and presence of the predator, respectively) than in the S form (from 48.3 to 64.1%). Thrashing at the water surface exposed larvae to a significantly greater risk of predation by the notonectid (P < 0.01), whereas predation occurred significantly less often when larvae were at the container wall (P < 0.05) and might reflect predator vigilance.ConclusionsBehavioural differences between larvae of the M and S form of An. gambiae in response to an acute predation risk is likely to be a reflection of different trade-offs between foraging and predator vigilance that might be of adaptive value in contrasting aquatic ecosystems. Future studies should explore the relevance of these findings under the wide range of natural settings where both forms co-exist in Africa.

Composition of Human Skin Microbiota Affects Attractiveness to Malaria Mosquitoes

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases.

Differential Attraction of Malaria Mosquitoes to Volatile Blends Produced by Human Skin Bacteria

The malaria mosquito Anopheles gambiae sensu stricto is mainly guided by human odour components to find its blood host. Skin bacteria play an important role in the production of human body odour and when grown in vitro, skin bacteria produce volatiles that are attractive to A. gambiae. The role of single skin bacterial species in the production of volatiles that mediate the host-seeking behaviour of mosquitoes has remained largely unknown and is the subject of the present study. Headspace samples were taken to identify volatiles that mediate this behaviour. These volatiles could be used as mosquito attractants or repellents. Five commonly occurring species of skin bacteria were tested in an olfactometer for the production of volatiles that attract A. gambiae. Odour blends produced by some bacterial species were more attractive than blends produced by other species. In contrast to odours from the other bacterial species tested, odours produced by Pseudomonas aeruginosa were not attractive to A. gambiae. Headspace analysis of bacterial volatiles in combination with behavioural assays led to the identification of six compounds that elicited a behavioural effect in A. gambiae. Our results provide, to our knowledge, the first evidence for a role of selected bacterial species, common on the human skin, in determining the attractiveness of humans to malaria mosquitoes. This information will be used in the further development of a blend of semiochemicals for the manipulation of mosquito behaviour.

Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases

New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 µM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast (∼30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.

The effect of aliphatic carboxylic acids on olfaction-based host-seeking of the malaria mosquito Anopheles gambiae sensu stricto.

The role of aliphatic carboxylic acids in host-seeking response of the malaria mosquito Anopheles gambiae sensu stricto was examined both in a dual-choice olfactometer and with indoor traps. A basic attractive blend of ammonia + lactic acid served as internal standard odor. Single carboxylic acids were tested in a tripartite blend with ammonia + lactic acid. Four different airflow stream rates (0.5, 5, 50, and 100 ml/min) carrying the compounds were tested for their effect on trap entry response in the olfactometer. In the olfactometer, propanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, heptanoic acid, octanoic acid, and tetradecanoic acid increased attraction relative to the basic blend. While several carboxylic acids were attractive only at one or two flow rates, tetradecanoic acid was attractive at all flow rates tested. Heptanoic acid was attractive at the lowest flow rate (0.5 ml/min), but repellent at 5 and 50 ml/min. Mixing the air stream laden with these 7 carboxylic acids together with the headspace of the basic blend increased attraction in two quantitative compositions. Subtraction of single acids from the most attractive blend revealed that 3-methylbutanoic acid had a negative effect on trap entry response. In the absence of tetradecanoic acid, the blend was repellent. In assays with MM-X traps, both a blend of 7 carboxylic acids + ammonia + lactic acid (all applied from low density polyethylene-sachets) and a simple blend of ammonia + lactic acid + tetradecanoic acid were attractive. The results show that carboxylic acids play an essential role in the host-seeking behavior of An. gambiae, and that the contribution to blend attractiveness depends on the specific compound studied.Electronic supplementary materialThe online version of this article (doi:10.1007/s10886-009-9668-7) contains supplementary material, which is available to authorized users.

Effect of human odours and positioning of CO2 release point on trap catches of the malaria mosquito Anopheles gambiae sensu stricto in an olfactometer

Abstract The anthropophilic malaria mosquito Anopheles gambiae sensu stricto responds to CO2 and human skin emanations. How these odorants affect the behaviour of this mosquito species is studied in an olfactometer. Carbon dioxide is released either as an homogeneous plume or in a turbulent fashion at two different positions from the trap entrance. Anopheles gambiae is deterred from entering a trap with CO2 as the only kairomone, when presented as an homogeneous or turbulent plume. This effect is completely overcome by the addition of skin emanations to the CO2 plume, with a high proportion of mosquitoes found in the trap with skin emanations. Rearrangement of the position of the turbulent CO2 source so that it is 5 cm downwind of the trap entrance overcomes the deterrent effect of CO2. Carbon dioxide alone, however, does not elicit higher proportions caught compared with clean air. Further studies with the CO2 source positioned 5 cm downwind of the trap entrance show that skin emanations alone result in fewer mosquitoes entering the trap than CO2+ skin emanations. Skin emanations induce more mosquitoes to fly into a trap than a synthetic blend of NH3+ l‐lactic acid when both are combined with CO2. It is concluded that CO2 is a poor kairomone when offered alone and that its presence in the plume at the trap entrance deters mosquitoes from entering. By contrast, when positioned just downwind of the trap entrance, CO2 appears to guide mosquitoes to the vicinity of the trap, where skin emanations then become the principle attractant, causing the mosquito trap entry response. The results of the study have implications for the design of odour‐baited traps for this mosquito species.

Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.

Synergism between ammonia, lactic acid and carboxylic acids as kairomones in the host-seeking behaviour of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae)

Host odours play a major role in the orientation and host location of blood-feeding mosquitoes. Anopheles gambiae Giles sensu stricto, which is the most important malaria vector in Africa, is a highly anthropophilic mosquito species, and the host-seeking behaviour of the females of this mosquito is guided by volatiles of human origin. Ammonia, lactic acid and several carboxylic acids are known to be present in the human odour blend. We investigated the effect of these compounds on naive female mosquitoes using a dual-port olfactometer. Ammonia was an attractant on its own, whereas lactic acid was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acids did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend. Our findings indicate that An. gambiae s.s. relies on the combination of ammonia, lactic acid and carboxylic acids in its orientation to human hosts. The role of lactic acid in this tripartite synergism differs from that reported for the yellow fever mosquito Aedes aegypti.

A Cluster of Candidate Odorant Receptors from the Malaria Vector Mosquito, Anopheles gambiae

Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.

Incubated human sweat but not fresh sweat attracts the malaria mosquito Anopheles gambiae sensu stricto

A dual-port olfactometer was used to quantify behavioral responses of the malaria mosquito Anopheles gambiae sensu stricto to volatiles emitted by sweat samples collected from three human volunteers. Significant attraction (P < 0.05) was found to sweat of two volunteers after two days and to one of the volunteers' sweat after one day of incubation at 37°C. There was no attraction to any of the fresh sweat samples. The pH of the sweat that became attractive after one or two days had changed from acidic (5.5–5.75) to alkaline (8.25–8.75), while the pH of the sweat of the third volunteer (pH 7) did not change during incubation. Microorganisms, present in all fresh sweat samples, showed distinct growth during incubation. The concentration of lactic acid decreased an average of 23% in two days of incubation. The role of the pH, skin microflora, and lactic acid in the differential attractiveness of human sweat samples is discussed.