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.

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.

Odor-mediated response of gravid Aedes aegypti to mosquito-associated symbiotic bacteria.

Complex oviposition decisions allow gravid Aedes aegypti mosquitoes to select suitable sites for egg-laying to increase the probability that their progeny will thrive. The bacterial communities present in larval niches influence mosquito oviposition behavior, and gravid mosquitoes transmit key microbial associates to breeding sites during oviposition. Our study evaluated whether symbiotic Klebsiella sp., which are strongly associated with mosquitoes, emit volatiles that affect mosquito oviposition decisions. Dual-choice behavioral assays demonstrated that volatile organic compounds emitted by Klebsiella sp. induce a preference in oviposition decisions by Ae. aegypti. Bacterial headspace volatiles were sampled by solid-phase microextraction, and subsequent combined gas chromatography and electroantennogram detection analysis, revealed that the antennae of gravid females detect two compounds present in the Klebsiella sp. headspace. These compounds were identified by gas chromatography and mass spectrometry as 2-ethyl hexanol and 2,4-di­tert-butylphenol. The binary blend of these compounds elicited a dose-dependent egg-laying preference by gravid mosquitoes. We propose that bacterial symbionts, which are associated with gravid mosquitoes and may be transferred to aquatic habitats during egg-laying, together with their volatiles act as oviposition cues indicating the suitability of active breeding sites to conspecific females.

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.

Seasonal variation in abundance and blood meal sources of primary and secondary malaria vectors within Kilombero Valley, Southern Tanzania.

BACKGROUND: Existing control tools have significantly reduced malaria over the past two decades. However, progress has been stalled due to increased resistance in primary vectors and the increasing role of secondary vectors. This study aimed to investigate the impact of seasonal change on primary and secondary vector abundance and host preference. Understanding the impact of seasonal dynamics of primary and secondary vectors on disease transmission will inform effective strategies for vector management and control. METHODS: Vector abundance was measured through longitudinal collection of mosquitoes, conducted monthly during the wet and dry seasons, in Sagamaganga, a village in the Kilombero Valley, Tanzania. Mosquitoes were collected indoors using CDC light traps and backpack aspirators, and outdoors using resting buckets baited with cattle urine. In addition, a direct measure of host preference was taken monthly using human- and cattle-baited mosquito electrocuting traps. A host census was conducted to provide an indirect measure of host preference together with monthly blood meal source analysis. All collected mosquitoes were assayed for Plasmodium sporozoites. RESULTS: A total of 2828 anophelines were collected, of which 78.5% and 21.4%, were primary and secondary vectors, respectively. The abundance of the primary vectors, Anopheles arabiensis and Anopheles funestus, and of the secondary vectors varied seasonally. Indirect measures of host preference indicated that all vectors varied blood meal choice seasonally, with the direct measure confirming this for An. arabiensis. All anopheline mosquitoes tested negative for sporozoites. CONCLUSIONS: At the study location, the abundance of both primary and secondary vectors changed seasonally. Indirect and direct measures of host preference demonstrated that An. arabiensis varied from being zoophilic to being more opportunistic during the wet and dry seasons. A similar trend was observed for the other vectors.

Mosquito host-seeking diel rhythm and chemosensory gene expression is affected by age and Plasmodium stages.

Malaria parasites can affect vector-related behaviours, increasing transmission success. Using Anopheles gambiae and Plasmodium falciparum, we consider the effect of interaction between infection stage and vector age on diel locomotion in response to human odour and the expression of antennal chemosensory genes. We identified age-dependent behavioural diel compartmentalisation by uninfected females post-blood meal. Infection disrupts overall and diel activity patterns compared with age-matched controls. In this study, mosquitoes carrying transmissible sporozoites were more active, shifting activity periods which corresponded with human host availability, in response to human odour. Older, uninfected, blood-fed females displayed reduced activity during their peak host-seeking period in response to human odour. Age- and infection stage-specific changes in odour-mediated locomotion coincide with altered transcript abundance of select chemosensory genes suggesting a possible molecular mechanism regulating the behaviour. We hypothesize that vector-related behaviours of female mosquitoes are altered by infection stage and further modulated by the age post-blood meal of the vector. Findings may have important implications for malaria transmission and disease dynamics.

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.

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.

Mosquito Host Seeking in 3D Using a Versatile Climate-Controlled Wind Tunnel System.

Future anthropogenic climate change is predicted to impact sensory-driven behaviors. Building on recent improvements in computational power and tracking technology, we have developed a versatile climate-controlled wind tunnel system, in which to study the effect of climate parameters, including temperature, precipitation, and elevated greenhouse gas levels, on odor-mediated behaviors in insects. To establish a baseline for future studies, we here analyzed the host-seeking behavior of the major malaria vector mosquito, Anopheles gambiae sensu strico, to human odor and carbon dioxide (CO2), under tightly controlled climatic conditions, and isolated from potential background contamination by the presence of an experimenter. When presented with a combination of human foot odor and CO2 (case study I), mosquitoes engaged in faster crosswind flight, spent more time in the filamentous odor plume and targeted the odor source more successfully. In contrast, female An. gambiae s. s. presented with different concentrations of CO2 alone, did not display host-seeking behavior (case study II). These observations support previous findings on the role of human host-associated cues in host seeking and confirm the role of CO2 as a synergist, but not a host-seeking cue on its own. Future studies are aimed at investigating the effect of climate change on odor-mediated behavior in mosquitoes and other insects. Moreover, the system will be used to investigate detection and processing of olfactory information in various behavioral contexts, by providing a fine-scale analysis of flight behavior.

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.

Hold your breath - Differential behavioral and sensory acuity of mosquitoes to acetone and carbon dioxide.

Host seeking in the yellow fever mosquito, Aedes aegypti, and the African malaria mosquito, Anopheles coluzzii, relies on specific and generic host-derived odorants. Previous analyses indicate that the behavioral response of these species depends differentially on the presence of carbon dioxide (CO2) and other constituents in human breath for activation and attraction. In this study, we use a flight tube assay and electrophysiological analysis to assess the role of acetone, a major component of exhaled human breath, in modulating the behavioral and sensory neuronal response of these mosquito species, in the presence and absence of CO2. When presented alone at ecologically relevant concentrations, acetone increases attraction in Ae. aegypti, but not in An. coluzzii. Moreover, in combination with CO2, human breath-equivalents of acetone ranging between 0.1 and 10 ppm reproduces a behavioral response similar to that observed to human breath in host-seeking Ae. aegypti, but not in An. coluzzii. Acetone does, however, reduce attraction to CO2 in An. coluzzii, when presented at a higher concentration of 10 ppm. We identify the capitate peg A neuron of the maxillary palp of both species as a dual detector of CO2 and acetone. The sensory response to acetone, or binary blends of acetone and CO2, reflects the observed behavioral output in both Ae. aegypti and An. coluzzii. We conclude that host recognition is contextual and dependent on a combination of ecologically relevant odorants at naturally occurring concentrations that are encoded, in this case, by differences in the temporal structure of the neuronal response. This information should be considered when designing synthetic blends for that optimally attract mosquitoes for monitoring and control.

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.

Ecological and Phylogenetic Relationships Shape the Peripheral Olfactory Systems of Highly Specialized Gall Midges (Cecidomiiydae).

Insects use sensitive olfactory systems to detect relevant host volatiles and avoid unsuitable hosts in a complex environmental odor landscape. Insects with short lifespans, such as gall midges (Diptera: Cecidomyiidae), are under strong selection pressure to detect and locate suitable hosts for their offspring in a short period of time. Ephemeral gall midges constitute excellent models for investigating the role of olfaction in host choice, host shift, and speciation. Midges mate near their site of emergence and females migrate in order to locate hosts for oviposition, thus females are expected to be more responsive to olfactory cues emitted by the host compared to males. In this study, we explored the correlation between host choice and the function of the peripheral olfactory system in 12 species of gall midges, including species with close phylogenetic relationships that use widely different host plants and more distantly related gall midge species that use similar hosts. We tested the antennal responses of males and females of the 12 species to a blend of 45 known insect attractants using coupled gas chromatographic-electroantennographic detection. When the species-specific response profiles of the gall midges were compared to a newly generated molecular-based phylogeny, we found they responded to the compounds in a sex- and species-specific manner. We found the physiological response profiles of species that use annual host plants, and thus have to locate their host every season, are similar for species with similar hosts despite large phylogenetic distances. In addition, we found closely related species with perennial hosts demonstrated odor response profiles that were consistent with their phylogenetic history. The ecology of the gall midges affects the tuning of the peripheral olfactory system, which in turn demonstrates a correlation between olfaction and speciation in the context of host use.

Functional characterization of mosquito short neuropeptide F receptors.

Mosquito blood feeding transiently inhibits sugar- and host seeking through neuropeptide signaling. Short neuropeptide F (sNPF) is one of the neuromodulators involved in this regulation. Here, we identified the genes for the sNPF precursor and the sNPF receptor in the southern house mosquito, Culex quinquefasciatus. Comparative analyses are made with the genes of the sNPF precursor and receptor from two other important vectors, Aedes aegypti and Anopheles coluzzii. We functionally characterized the receptors in all three species using endogenous neuropeptides, and quantified their transcript expression following a blood meal and a sugar meal. Our analysis reveals several Cx. quinquefasciatus-specific duplications of the sNPF-3 isoform on the sNPF precursor, which are not reflected in the precursors of the other two species. In contrast, the structure of the sNPF receptors is highly conserved within mosquitoes, and a putative ligand binding region is proposed and discussed. Reflecting the high structural conservation, the sNPF receptor sensitivity to endogenous sNPF isoforms is conserved across mosquito species. Using quantitative real time PCR, we demonstrate that transcript abundance of the sNPF receptor and precursor is regulated following feeding, only in Cx. quinquefasciatus. We discuss our findings in relation to previous work on sNPF signaling and its role in feeding regulation.

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.

Functional characterization of the dual allatostatin-A receptors in mosquitoes.

The neuropeptide allatostatin-A (AstA) and its cognate receptors (AstARs) are involved in the modulation of feeding behavior, which in hematophagous insects includes the regulation of the disease vector-related behaviors, host seeking and blood feeding. In mosquitoes and other dipterans, there are two copies of AstAR, contrasting with the single copy found in other insects. In this study, we identified and cloned the dual AstAR system of two important disease vectors Aedes aegypti and Culex quinquefasciatus, and compared them with those previously described, including those in Anopheles coluzzii and Drosophila melanogaster. Phylogenetic analysis of the AstARs revealed that the mosquito AstAR1s has retained a similar amino acid sequence as the AstARs from non-dipteran insect species. Intron analysis revealed that the number of introns accumulated in the AstAR2s is similar to that in other insects, and that introns are conserved within the receptor types, but that only the final two introns are conserved across AstAR1s and 2s. We functionally characterized the dual AstARs in An. coluzzii, Ae. aegypti and Cx. quinquefasciatus by stably expressing the receptors in a Chinese hamster oocyte cell line (CHO) also stably expressing a promiscuous G-protein (G16), and challenged them with the endogenous isoforms of AstA from the three mosquito species. In the culicine mosquitoes, Ae. aegypti and Cx. quinquefasciatus, the AstARs demonstrated differential sensitivity to AstA, with the AstAR2s displaying a higher sensitivity than the AstAR1s, suggesting a divergence of functional roles for these AstARs. In contrast, both An. coluzzii AstARs demonstrated a similar sensitivity to the AstA ligands. We discuss our findings in the light of AstA acting as a regulator of blood feeding in mosquitoes. A better understanding of the regulation of host seeking and blood feeding in vector mosquitoes will lead to the rational development of novel approaches for vector control.

Grass Pollen Affects Survival and Development of Larval Anopheles arabiensis (Diptera: Culicidae).

Nutrients in breeding sites are critical for the survival and development of malaria mosquitoes, having a direct impact on vectorial capacity. Yet, there is a limited understanding about the natural larval diet and its impact on the individual fitness of mosquitoes. Recent studies have shown that gravid Anopheles arabiensis Patton (Diptera: Culicidae) are attracted by and oviposit in grass-associated habitats. The pollen provided by these grasses is a potential source of nutrients for the larvae. Here, we assess the effect of Typha latifolia L. (Poales: Typhaceae), Echinochloa pyramidalis Lamarck, Pennisetum setaceum Forsskål, and Zea mays L. pollen on larval survival and rate of development in An. arabiensis under laboratory conditions. In addition, we characterize the carbon to nitrogen ratio and the size of pollen grains as a measure of diet quality. Carbon-rich pollen with a small grain size (T. latifolia and P. setaceum; 9.7 ± 0.3 × 103 and 5.5 ± 0.2 × 104 µm3, respectively) resulted in enhanced rates of development of An. arabiensis. In contrast, the larva fed on the nitrogen-rich control diet (TetraMin) was slower to develop, but demonstrated the highest larval survival. Larvae fed on carbon-rich and large-grained Z. mays pollen (4.1 ± 0.2 × 105 µm3) survived at similar levels as those fed on the control diet and also took a longer time to develop compared with larvae fed on the other pollens. While males and females did not appear to develop differently on the different pollen diets, males consistently emerged faster than their female counterparts. These results are discussed in relation to integrated vector management.

A key malaria metabolite modulates vector blood seeking, feeding, and susceptibility to infection.

Malaria infection renders humans more attractive to Anopheles gambiae sensu lato mosquitoes than uninfected people. The mechanisms remain unknown. We found that an isoprenoid precursor produced by Plasmodium falciparum, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), affects A. gambiae s.l. blood meal seeking and feeding behaviors as well as susceptibility to infection. HMBPP acts indirectly by triggering human red blood cells to increase the release of CO2, aldehydes, and monoterpenes, which together enhance vector attraction and stimulate vector feeding. When offered in a blood meal, HMBPP modulates neural, antimalarial, and oogenic gene transcription without affecting mosquito survival or fecundity; in a P. falciparum-infected blood meal, sporogony is increased.

The role of grass volatiles on oviposition site selection by Anopheles arabiensis and Anopheles coluzzii.

BACKGROUND: The reproductive success and population dynamics, of Anopheles malaria mosquitoes is strongly influenced by the oviposition site selection of gravid females. Mosquitoes select oviposition sites at different spatial scales, starting with selecting a habitat in which to search. This study utilizes the association of larval abundance in the field with natural breeding habitats, dominated by various types of wild grasses, as a proxy for oviposition site selection by gravid mosquitoes. Moreover, the role of olfactory cues emanating from these habitats in the attraction and oviposition stimulation of females was analysed. METHODS: The density of Anopheles larvae in breeding sites associated with Echinochloa pyramidalis, Echinochloa stagnina, Typha latifolia and Cyperus papyrus, was sampled and the larvae identified to species level. Headspace volatile extracts of the grasses were collected and used to assess behavioural attraction and oviposition stimulation of gravid Anopheles arabiensis and Anopheles coluzzii mosquitoes in wind tunnel and two-choice oviposition assays, respectively. The ability of the mosquitoes to differentiate among the grass volatile extracts was tested in multi-choice tent assays. RESULTS: Anopheles arabiensis larvae were the most abundant species found in the various grass-associated habitats. The larval densities described a hierarchical distribution, with Poaceae (Echinochloa pyramidalis and Echinochloa stagnina)-associated habitat sites demonstrating higher densities than that of Typha-associated sites, and where larvae were absent from Cyperus-associated sites. This hierarchy was maintained by gravid An. arabiensis and An. coluzzii mosquitoes in attraction, oviposition and multi-choice assays to grass volatile extracts. CONCLUSIONS: The demonstrated hierarchical preference of gravid An. coluzzii and An. arabiensis for grass volatiles indicates that vegetation cues associated with larval habitats are instrumental in the oviposition site choice of the malaria mosquitoes. Identifying volatile cues from grasses that modulate gravid malaria mosquito behaviours has distinct potential for the development of tools to be used in future monitoring and control methods.

A(maize)ing attraction: gravid Anopheles arabiensis are attracted and oviposit in response to maize pollen odours.

BACKGROUND: Maize cultivation contributes to the prevalence of malaria mosquitoes and exacerbates malaria transmission in sub-Saharan Africa. The pollen from maize serves as an important larval food source for Anopheles mosquitoes, and females that are able to detect breeding sites where maize pollen is abundant may provide their offspring with selective advantages. Anopheles mosquitoes are hypothesized to locate, discriminate among, and select such sites using olfactory cues, and that synthetic volatile blends can mimic these olfactory-guided behaviours. METHODS: Two-port olfactometer and two-choice oviposition assays were used to assess the attraction and oviposition preference of gravid Anopheles arabiensis to the headspace of the pollen from two maize cultivars (BH-660 and ZM-521). Bioactive compounds were identified using combined gas chromatography and electroantennographic detection from the headspace of the cultivar found to be most attractive (BH-660). Synthetic blends of the volatile compounds were then assessed for attraction and oviposition preference of gravid An. arabiensis, as above. RESULTS: Here the collected headspace volatiles from the pollen of two maize cultivars was shown to differentially attract and stimulate oviposition in gravid An. arabiensis. Furthermore, a five-component synthetic maize pollen odour blend was identified, which elicited the full oviposition behavioural repertoire of the gravid mosquitoes. CONCLUSIONS: The cues identified from maize pollen provide important substrates for the development of novel control measures that modulate gravid female behaviour. Such measures are irrespective of indoor or outdoor feeding and resting patterns, thus providing a much-needed addition to the arsenal of tools that currently target indoor biting mosquitoes.