Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

Mosquito brains encode unique features of human odour to drive host seeking.

A globally invasive form of the mosquito Aedes aegypti specializes in biting humans, making it an efficient disease vector1. Host-seeking female mosquitoes strongly prefer human odour over the odour of animals2,3, but exactly how they distinguish between the two is not known. Vertebrate odours are complex blends of volatile chemicals with many shared components4-7, making discrimination an interesting sensory coding challenge. Here we show that human and animal odours evoke activity in distinct combinations of olfactory glomeruli within the Ae. aegypti antennal lobe. One glomerulus in particular is strongly activated by human odour but responds weakly, or not at all, to animal odour. This human-sensitive glomerulus is selectively tuned to the long-chain aldehydes decanal and undecanal, which we show are consistently enriched in human odour and which probably originate from unique human skin lipids. Using synthetic blends, we further demonstrate that signalling in the human-sensitive glomerulus significantly enhances long-range host-seeking behaviour in a wind tunnel, recapitulating preference for human over animal odours. Our research suggests that animal brains may distil complex odour stimuli of innate biological relevance into simple neural codes and reveals targets for the design of next-generation mosquito-control strategies.

Mosquito odour-baited mass trapping reduced malaria transmission intensity: a result from a controlled before-and-after intervention study.

BACKGROUND: Conventional vector control strategies have significantly reduced the malaria burden. The sustainability of these methods is currently challenged. Odour-based traps are emerging technologies that can complement the existing tools. Implementation of odour-based traps for mass trapping is limited due to the restricted range of vectors caught with available carbon dioxide-dependent lures, and the lack of comprehensive field studies. The objective of this study was to assess the impact of odour-mediated mass trapping targeting outdoor vectors, using a synthetic cattle urine lure that attracts a wide range of vector species in a variety of physiological states, on malaria prevalence and entomological parameters to determine malaria transmission intensities. METHODS: A controlled before-and-after study was conducted in two rural communities in southern Ethiopia. Baseline monthly entomological and seasonal cross-sectional malaria prevalence surveys were conducted in both communities for a year. Then, mass trapping of mosquitoes was conducted in one of the villages, while the monthly entomological surveillance and seasonal malaria prevalence surveys continued in both villages. Generalised linear mixed models were constructed and tested to determine which factors were significantly affected by the intervention. RESULTS: Mass trapping contributed to the reduction of the population of the principal malaria vector, Anopheles arabiensis, and the associated entomological indicators, the human bite rate (HBR) and the entomological inoculation rate (EIR), in the intervention village compared to the control village. The intervention village had an average HBR by An. arabiensis of 3.0 (95% CI 1.4-4.6) during the peak malaria transmission season, compared to 10.5 (95% CI - 0.5-21.5; P < 0.0001) in the control village. The intervention village (mean 0.02, 95% CI - 0.05-0.4.8) had a daily EIR eight times lower than the control village (mean 0.17, 95% CI), which likely contributed to the reduced malaria prevalence in the intervention community following its introduction by ca. 60% (95% CI 55-63). CONCLUSIONS: The combined use of odour-based mass trapping and conventional control strategies coincided with a reduction of human-vector contact and malaria prevalence, providing support for odour-baited technologies as a viable option for next-generation vector control tools. Further cluster-randomised control studies are recommended in different eco-epidemiological settings with varying malaria transmission intensities.

Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii.

The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR-Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.

Distance from human dwellings differentially affects the efficacy of a synthetic cattle urine odour lure to trap malaria vectors.

BACKGROUND: Cost-effective outdoor-based devices for surveillance and control of outdoor mosquito vector populations can substantially improve their efficacy when baited with synthetic human and animal odours. This study aimed at assessing the dose-dependent efficacy of a previously developed synthetic cattle urine odour to lure malaria vectors, and other mosquito species, to traps placed at different distances from human dwellings outdoors. METHODS: The efficacy of the cattle urine odour lure was assessed through a 5 × 5 Latin square design, using two sets of 5 Suna traps placed at either 1.5 m or 5 m from an adjacent human dwelling, in the rural village of Sagamaganga, Tanzania. Each trap was deployed with one of four doses of the synthetic cattle urine odour blend or a solvent control (heptane). Traps were rotated daily so that each dose and control visited each position twice over a period of 20 experimental nights. The relative attractiveness of each treatment dose and control was compared using a generalized linear mixed model for each species caught. RESULTS: A total of 1568 mosquitoes were caught, of which 783 were anophelines and 785 were culicines. Of the anophelines, 41.6 and 58.3% were primary and secondary vector species, respectively. Unfed and fed females of the primary vector, Anopheles arabiensis, were caught dose-dependently, close to human dwellings (1.5 m), whereas unfed, fed and gravid secondary vector Anopheles pharoensis females were caught dose-dependently, but at a farther distance from the dwellings (5 m). Females of Culex spp. were caught dose-dependently in similar numbers irrespective of the distance from human dwellings. CONCLUSIONS: This study further clarifies the factors to be considered for the implementation of outdoor trapping using the synthetic cattle urine lure to target exophilic and exophagic malaria vectors, for which efficient surveillance and control tools are currently lacking. The findings resulting from this study make significant progress in providing the needed information to overcome the regulatory obstacles to make this tool available for integrated vector management programs, including registration, as well as evaluation and regulation by the World Health Organization.

Effect of non-human hosts on the human biting rate of primary and secondary malaria vectors in Tanzania.

BACKGROUND: Malaria vectors vary in feeding preference depending on their innate behaviour, host availability and abundance. Host preference and human biting rate in malaria vectors are key factors in establishing zooprophylaxis and zoopotentiation. This study aimed at assessing the impact of non-human hosts in close proximity to humans on the human biting rate of primary and secondary malaria vectors, with varying host preferences. METHODS: The effect of the presence of non-human hosts in close proximity to the human host on the mean catches per person per night, as a proxy for mosquito biting rate, was measured using mosquito-electrocuting traps (METs), in Sagamaganga, Kilombero Valley, Tanzania. Two experiments were designed: (1) a human versus a calf, each enclosed in a MET, and (2) a human surrounded by three calves versus a human alone, with each human volunteer enclosed individually in a MET spaced 10 m apart. Each experiment was conducted on alternate days and lasted for 36 nights per experiment. During each experiment, the positions of hosts were exchanged daily (except the human in experiment 2). All anopheline mosquitoes caught were assayed for Plasmodium sporozoites using enzyme-linked immunosorbent assay. RESULTS: A total of 20,574 mosquitoes were captured and identified during the study, of which 3608 were anophelines (84.4% primary and 15.6% secondary malaria vectors) and 17,146 were culicines. In experiment 1, the primary malaria vector, Anopheles arabiensis, along with Culex spp. demonstrated a preference for cattle, while the primary vectors, Anopheles funestus, preferred humans. In experiment 2, both primary vectors, An. arabiensis and An. funestus, as well as the secondary vector Anopheles rivolurum, demonstrated behaviours amenable to zooprophylaxis, whereas Culex spp. increased their attraction to humans in the presence of nearby cattle. All anopheline mosquitoes tested negative for sporozoites. CONCLUSIONS: The findings of this study provide support for the zooprophylaxis model for malaria vectors present in the Kilombero Valley, and for the zoopotentiation model, as it pertains to the Culex spp. in the region. However, the factors regulating zooprophylaxis and zoopotentiation are complex, with different species-dependent mechanisms regulating these behaviours, that need to be considered when designing integrated vector management programmes.

Fermenting molasses and a synthetic odour blend to attract blood-fed Anopheles coluzzii.

Collecting blood-fed mosquitoes to monitor pathogen presence or to gather information on the host blood meal is often challenging. Fermenting molasses can be used to produce carbon dioxide to attract host-seeking mosquitoes, however, earlier work indicated that it may also attract blood-fed mosquitoes in the field. In the current study, these field results were validated in an experimental setting using a large cage setup with Anopheles coluzzii (Diptera, Culicidae). Blood-fed mosquitoes were indeed attracted to fermenting molasses with the highest attraction at 72 hours post feeding, which was used for subsequent experiments. Next, it was tested if fermentation of molasses is required for attraction, and whether it acts as an oviposition attractant, increases egg laying, or increases mosquito survival. The compounds that could be responsible for attraction were identified by combined electrophysiology and chemical analyses and formulated into a synthetic blend. Fermenting molasses attracted blood-fed mosquitoes in the large cage study, while fermenting sugar and non-fermenting molasses did not. The fecundity of blood-fed mosquitoes increased after feeding on fermenting molasses, however, compounds emanating from molasses did not trigger oviposition. The synthetic blend attracted blood-fed mosquitoes and may be used to determine mosquito host selection and for xenomonitoring, as 'flying syringes' to detect non-vector borne pathogens.

Malaria mosquitoes acquire and allocate cattle urine to enhance life history traits.

BACKGROUND: Nutrient acquisition and allocation integrate foraging and life-history traits in insects. To compensate for the lack of a particular nutrient at different life stages, insects may acquire these through supplementary feeding, for example, on vertebrate secretions, in a process known as puddling. The mosquito Anopheles arabiensis emerges undernourished, and as such, requires nutrients for both metabolism and reproduction. The purpose of this study was to assess whether An. arabiensis engage in puddling on cattle urine to obtain nutrients to improve life history traits. METHODS: To determine whether An. arabiensis are attracted to the odour of fresh, 24 h, 72 h and 168 h aged cattle urine, host-seeking and blood-fed (48 h post-blood meal) females were assayed in a Y-tube olfactometer, and gravid females assessed in an oviposition assay. Combined chemical and electrophysiological analyses were subsequently used to identify the bioactive compounds in all four age classes of cattle urine. Synthetic blends of bioactive compounds were evaluated in both Y-tube and field assays. To investigate the cattle urine, and its main nitrogenous compound, urea, as a potential supplementary diet for malaria vectors, feeding parameters and life history traits were measured. The proportion of female mosquitoes and the amount of cattle urine and urea imbibed, were assessed. Following feeding, females were evaluated for survival, tethered flight and reproduction. RESULTS: Host-seeking and blood-fed An. arabiensis were attracted to the natural and synthetic odour of fresh and aged cattle urine in both laboratory and field studies. Gravid females were indifferent in their response to cattle urine presence at oviposition sites. Host-seeking and blood-fed females actively imbibed cattle urine and urea, and allocated these resources according to life history trade-offs to flight, survival or reproduction, as a function of physiological state. CONCLUSIONS: Anopheles arabiensis acquire and allocate cattle urine to improve life history traits. Supplementary feeding on cattle urine affects vectorial capacity directly by increasing daily survival and vector density, as well as indirectly by altering flight activity, and thus should be considered in future models.

Cattle-Derived Unsaturated Aldehydes Repel Biting Midges and Mosquitoes.

Host-derived repellents offer a novel way to reduce disease vector-host interactions, particularly for vectors and nuisance pests where commercial repellents are not available, e.g., Culicoides biting midges. By revising the criteria previously used to identify bioactive volatile organic compounds for Culicoides nubeculosus, we identify (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, and (E)-2-nonenal to be differentially present in the headspace odour of cattle hair and to elicit antennal responses in this research model species. A blend of these unsaturated aldehydes elicited an aversive response in C. nubeculosus, as well as a repellent response in three disease vector mosquito species, Aedes aegypti, Culex quinquefasciatus and Anopheles coluzzii, a response that was stronger than that to the commercially available repellents tested (DEET, IR3535, PMD, icaridin, and d-allethrin). Culicoides nubeculosus was behaviourally indifferent to these commercially available repellents tested, except d-allethrin to which it was attracted. The identification of a host-derived repellent odour blend, which reduces the interaction between biting midges extends the array of tools to be used in integrated vector management of these and other disease vectors.

De novo transcriptome sequencing of the northern fowl mite, Ornithonyssus sylviarum, shed light on parasitiform poultry mites evolution and its chemoreceptor repertoires.

The northern fowl mite (NFM), Ornithonyssus sylviarum, and the poultry red mite (PRM), Dermanyssus gallinae, are the most serious pests of poultry, both of which have an expanding global prevalence. Research on NFM has been constrained by a lack of genomic and transcriptomic data. Here, we report and analyze the first global transcriptome data across all mite live stages and sexes. A total of 28,999 unigenes were assembled, of which 19,750 (68.10%) were annotated using seven functional databases. The biological function of these unigenes was classified using the GO, KOG, and KEGG databases. To gain insight into the chemosensory receptor-based system of parasitiform mites, we furthermore assessed the gene repertoire of gustatory receptors (GRs) and ionotropic receptors (IRs), both of which encode putative ligand-gated ion channel proteins. While these receptors are well characterized in insect model species, our understanding of chemosensory detection in mites and ticks is in its infancy. To address this paucity of data, we identified 9 IR/iGluRs and 2 GRs genes by analyzing transcriptome data in the NFM, while 9 GRs and 41 IR/iGluRs genes were annotated in the PRM genome. Taken together, the transcriptomic and genomic annotation of these two species provide a valuable reference for studies of parasitiform mites and also help to understand how chemosensory gene family expansion/contraction events may have been reshaped by an obligate parasitic lifestyle compared with their free-living closest relatives. Future studies should include additional species to validate this observation and functional characterization of the identified proteins as a step forward in identifying tools for controlling these poultry pests.

Regulation of the antennal transcriptome of the dengue vector, Aedes aegypti, during the first gonotrophic cycle.

BACKGROUND: In the light of dengue being the fastest growing transmissible disease, there is a dire need to identify the mechanisms regulating the behaviour of the main vector Aedes aegypti. Disease transmission requires the female mosquito to acquire the pathogen from a blood meal during one gonotrophic cycle, and to pass it on in the next, and the capacity of the vector to maintain the disease relies on a sustained mosquito population. RESULTS: Using a comprehensive transcriptomic approach, we provide insight into the regulation of the odour-mediated host- and oviposition-seeking behaviours throughout the first gonotrophic cycle. We provide clear evidence that the age and state of the female affects antennal transcription differentially. Notably, the temporal- and state-dependent patterns of differential transcript abundance of chemosensory and neuromodulatory genes extends across families, and appears to be linked to concerted differential modulation by subsets of transcription factors. CONCLUSIONS: By identifying these regulatory pathways, we provide a substrate for future studies targeting subsets of genes across disparate families involved in generating key vector behaviours, with the goal to develop novel vector control tools.

Modulation of odour-guided behaviour in mosquitoes.

Mosquitoes are emerging as model systems with which to study innate behaviours through neuroethology and functional genomics. Decades of work on these disease vectors have provided a solid behavioural framework describing the distinct repertoire of predominantly odour-mediated behaviours of female mosquitoes, and their dependence on life stage (intrinsic factors) and environmental cues (extrinsic factors). The purpose of this review is to provide an overview of how intrinsic factors, including adult maturation, age, nutritional status, and infection, affect the attraction to plants and feeding on plant fluids, host seeking, blood feeding, supplemental feeding behaviours, pre-oviposition behaviour, and oviposition in female mosquitoes. With the technological advancements in the recent two decades, we have gained a better understanding of which volatile organic compounds are used by mosquitoes to recognise and discriminate among various fitness-enhancing resources, and characterised their neural and molecular correlates. In this review, we present the state of the art of the peripheral olfactory system as described by the neural physiology, functional genomics, and genetics underlying the demonstrated changes in the behavioural repertoire in female mosquitoes. The review is meant as a summary introduction to the current conceptual thinking in the field.

Correction: Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

[This corrects the article DOI: 10.1371/journal.pbio.1002454.].

Development of a chimeric odour blend for attracting gravid malaria vectors.

BACKGROUND: Odour-based tools targeting gravid malaria vectors may complement existing intervention strategies. Anopheles arabiensis are attracted to, and stimulated to oviposit by, natural and synthetic odours of wild and domesticated grasses associated with mosquito breeding sites. While such synthetic odour lures may be used for vector control, these may have limited efficacy when placed in direct competition with the natural source. In this study, workflows developed for plant-feeding pests was used to design and evaluate a chimeric odour blend based on shared attractive compounds found in domesticated grass odours. METHODS: Variants of a synthetic odour blend, composed of shared bioactive compounds previously identified in domesticated grasses, was evaluated sequentially in a two-choice olfactometer to identify a ratio-optimized attractive blend for malaria vectors. During this process, blends with ratios that were significantly more attractive than the previously identified synthetic rice blend were compared to determine which was most attractive in the two-choice olfactometer. To determine whether all volatile components of the most attractive blend were necessary for maximal attraction, subtractive assays were then conducted, in which individual components were removed for the most attractive blend, to define the final composition of the chimeric blend. Binary logistic regression models were used to determine significance in all two-choice assays. The chimeric blend was then assessed under field conditions in malaria endemic villages in Ethiopia, to assess the effect of dose, trap type, and placement relative to ground level. Field data were analyzed both descriptively and using a Welch-corrected t-test. RESULTS: A ratio-optimized chimeric blend was identified that significantly attracted gravid An. arabiensis under laboratory conditions. In the field, trap captures of An. arabiensis and Anopheles pharoensis were dependent on the presence of the lure, trap type (CDC, BG Sentinel and Suna traps), placement relevant to ground level, with low release rates generally luring more mosquitoes. CONCLUSIONS: The workflow designed for the development of chimeric lures provides an innovative strategy to target odour-mediated behaviours. The chimeric lure identified here can be used in existing trapping systems, and be customized to increase sustainability, in line with goals of the Global Vector Control Response Group.

Plasmodium falciparum gametocyte-induced volatiles enhance attraction of Anopheles mosquitoes in the field.

BACKGROUND: Plasmodium parasites manipulate the interaction between their mosquito and human hosts. Patients infected with gametocytes attract anopheline mosquitoes differentially compared to healthy individuals, an effect associated with an increased release of attractive volatile cues. This odour-driven manipulation is partly mediated by the gametocyte-specific metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which induces increased release of select aldehydes and terpenes from red blood cells and results in the enhanced attraction of host-seeking mosquitoes, which are vectors of malaria. This study investigates the effect of the HMBPP-induced volatiles on the attraction of wild Anopheles mosquitoes to humans under field conditions. METHODS: The efficacy of the HMBPP-induced odour blend to attract Anopheles was evaluated in a 4 × 6 Latin rectangular study design indoors using baited Suna traps. Furthermore, to assess the efficacy of the HMBPP-induced odour blend in (1) augmenting the attractiveness of human odour, and (2) attracting Anopheles mosquitoes in the absence of human odour, a two-choice assay using host decoy traps (HDTs) was used and evaluated using binomial generalized regression. RESULTS: Traps baited with the HMBPP-induced odour blend attracted and caught both Anopheles arabiensis and Anopheles pharoensis females in a dose-dependent manner in the presence of background human odour, up to 2.5 times that of an unbaited trap. Given a choice between human odour and human odour laden with the HMBPP-induced odour blend, mosquitoes differentially preferred traps augmented with the HMBPP-induced odour blend, which caught twice as many female An. arabiensis. Traps baited with the HMBPP-induced odour blend but lacking the background of human odour were not effective in attracting and catching mosquitoes. CONCLUSION: The findings of the present study revealed that the HMBPP-induced odour blend, when augmented with human body odour, is attractive to anopheline mosquitoes and could be used as a complementary vector control tool along with existing strategies.

Evolution of mosquito preference for humans linked to an odorant receptor.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly's high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.

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.

Shady business: understanding the spatial ecology of exophilic Anopheles mosquitoes.

BACKGROUND: Understanding the ecology of exophilic anophelines is a key step toward developing outdoor control strategies to complement existing indoor control tools against malaria vectors. This study was conducted to assess the movement pattern of exophilic Anopheles mosquitoes between blood meal sources and resting habitats, and the landscape factors dictating their resting habitat choice. RESULTS: Resting clay pots were placed at 5 m, 25 m, 50 m, 75 m and 100 m away from isolated focal houses, radiating from them in four directions. The locations of the clay pots represent heterogeneous land cover types at a relatively fine spatial scale in the landscape. The effect of the landscape characters on the number of both female and male anophelines caught was modelled using zero-inflated negative binomial regression with a log link function. A total of 420 Anopheles mosquitoes (353 females and 67 males) belonging to three species; Anopheles arabiensis, Anopheles pharoensis, and Anopheles tenebrosus were caught in the resting clay pots, with An. arabiensis being the dominant species. Canopy cover, distance from the house, and land cover type were the significant landscape characters influencing the aggregation of resting mosquitoes. Both the count and binary models showed that canopy cover was the strongest predictor variable on the counts and the presence of Anopheles mosquitoes in the clay pots. Female Anopheles were most frequently found resting in the pots placed in banana plantations, and at sampling points that were at the greater distances (75 m and 100 m) from the focal house. CONCLUSIONS: This study showed that exophilic Anopheles mosquitoes tend to rest in shaded areas some distance away from human habitation. These findings are important when targeting mosquitoes outdoors, complementing the existing effort being made to control malaria vectors indoors.

Sweet attraction: sugarcane pollen-associated volatiles attract gravid Anopheles arabiensis.

BACKGROUND: Anopheles arabiensis is a key vector for the transmission of human malaria in sub-Saharan Africa. Over the past 10,000 years, humans have successfully cultivated grasses and altered the landscape, creating An. arabiensis favourable environments that contain excellent habitats for both larvae and adults. Sugarcane is the most expanding agricultural system in sub-Saharan Africa, and is linked to the increased threat of malaria in rural communities. The prolific production and wind dispersal of sugarcane pollen, together with standing pools of water, often provide, as a result of irrigation, a nutrient-rich environment for the offspring of gravid malaria mosquitoes. RESULTS: In the present study, sugarcane pollen-associated volatiles from two cultivars are shown to attract gravid An. arabiensis in a still air two-port olfactometer and stimulate egg laying in an oviposition bioassay. Through combined gas chromatography and electroantennographic detection, as well as combined gas chromatography and mass spectrometric analyses, we identified the bioactive volatiles and generated a synthetic blend that reproduced the full behavioural repertoire of gravid mosquitoes in the Y-tube assay. Two subtractive odour blends, when compared with the full blend, were significantly more attractive. These three and four-component subtractive blends share the compounds (1R)-(+)-α-pinene, nonanal and benzaldehyde, of which, (1R)-(+)-α-pinene and nonanal are found in the attractive odour blends from rice plants and maize pollen. In pairwise comparisons, the rice synthetic odour blend was more attractive to gravid mosquitoes than either of the pollen blends, whereas the pollen blends did not differ in attraction. CONCLUSIONS: The attraction of gravid females to sugarcane pollen volatiles demonstrated in this study, together with the previously found grass-associated volatiles, raise the potential of developing a bioactive chimeric blend to attract gravid malaria mosquitoes. This is discussed in relation to the development of novel and cost-effective vector control measures.