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Background: The outbreaks of bluetongue and Schmallenberg disease in Europe have increased efforts to understand the ecology of Culicoides biting midges and their role in pathogen transmission. However, most studies have focused on a specific habitat, region, or country. To facilitate wider comparisons, and to obtain a better understanding of the spread of disease through Europe, the present study focused on monitoring biting midge species diversity in three different habitat types and three countries across Europe., Methods: Biting midges were trapped using Onderstepoort Veterinary Institute light traps at a total of 27 locations in Sweden, the Netherlands and Italy, comprising farm, peri-urban and wetland habitats. From July 2014 to June 2015 all locations were sampled monthly, except for during the winter months. Trapped midges were counted and identified morphologically. Indices on species richness, evenness and diversity were calculated. Community compositions were analysed using non-metric multidimensional scaling (NMDS) techniques., Results: A total of 50,085 female midges were trapped during 442 collection nights. More than 88% of these belonged to the Obsoletus group. The highest midge diversity was found in Sweden, while species richness was highest in the Netherlands, and most specimens were trapped in Italy. For habitats within countries, diversity of the trapped midges was lowest for farms in all countries. Differences in biting midge species communities were more distinct across the three countries than the three habitat types., Conclusions: A core midge community could be identified, in which the Obsoletus group was the most abundant. Variations in vector communities across countries imply different patterns of disease spread throughout Europe. How specific species and their associated communities affect disease risk is still unclear. Our results emphasize the importance of midge diversity data at community level, how this differs across large geographic range within Europe, and its implications on assessing risks of midge-borne disease outbreaks.

It has been suggested that Plasmodia manipulate their vertebrate hosts to enhance parasite transmission. Using a dual-choice olfactometer, we investigated the attraction of Anopheles gambiae to 50 Kenyan children (aged 5-12 years) who were naturally infected with Plasmodium falciparum or noninfected controls. Microscopic gametocyte carriers attracted almost 2 times more mosquitoes than children who were parasite free, harbored asexual stages, or had gametocytes at submicroscopic densities. By using highly sensitive stage-specific molecular methods to detect P. falciparum, we show that gametocytes-and not their noninfectious asexual progenitors-induce increased attractiveness of humans to mosquitoes. Our findings therefore support the parasite host manipulation hypothesis., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The deployment of odor-baited tools for sampling and controlling malaria vectors is limited by a lack of potent synthetic mosquito attractants. A synthetic mixture of chemical compounds referred to as "the Mbita blend" (MB) was shown to attract as many host-seeking malaria mosquitoes as attracted to human subjects. We hypothesized that this effect could be enhanced by adding one or more attractive compounds to the blend. We tested changes in the capability of MB (ammonia + L-lactic acid + tetradecanoic acid +3-methyl-1-butanol + carbon dioxide) to attract host-seeking malaria mosquitoes by addition of selected dilutions of butyl-2-methylbutanoate (1:10,000), 2-pentadecanone (1:100), 1-dodecanol (1:10,000), and butan-1-amine (1:10,000,000). The experiments were conducted in semi-field enclosures and in a village in western Kenya. In semi-field enclosures, the attraction of Anopheles gambiae sensu stricto females to MB-baited traps was not enhanced by adding butyl-2-methylbutanoate. There was, however, an increase in the proportion of An. gambiae caught in traps containing MB augmented with the selected dilutions of butan-1-amine, 2-pentadecanone, and 1-dodecanol. When tested in the village, addition of butan-1-amine to MB enhanced catches of female An. gambiae sensu lato, An. funestus, and Culex mosquitoes. 1-Dodecanol increased attraction of An. gambiae s.l. to the MB, while addition of 2-pentadecanone improved trap catches of An. funestus and Culex mosquitoes. This study demonstrates the possibility of enhancing synthetic odor blends for trapping the malarial mosquitoes An. gambiae s.l. and An. funestus, as well as some culicine species. The findings provide promising results for the optimization and utilization of synthetic attractants for sampling and controlling major disease vectors.

Background: Malaria mosquitoes often blood feed indoors on human hosts. The mosquitoes predominantly enter houses via open eaves. Host-seeking is odour-driven, and finding a host depends on the quality of the odour plume and whether the route towards the host is free of obstructions. Little is known about in-flight behaviour of mosquitoes during house entry. This semi-field study visualizes mosquito house entry in three dimensions (3D) and offers new insights for optimizing vector control interventions., Methods: The approach and house entry of Anopheles gambiae sensu stricto was studied in a semi-field set-up using video-recorded flight tracks and 3D analysis. Behavioural parameters of host-seeking female mosquitoes were visualized with respect to their position relative to the eave as well as whether a mosquito would enter or not. Host odour was standardized using an attractive synthetic blend in addition to CO2. The study was conducted in western Kenya at the Thomas Odhiambo Campus of the International Centre of Insect Physiology and Ecology, Mbita., Results: The majority of host-seeking An. gambiae approached a house with a flight altitude at eave level, arriving within a horizontal arc of 180°. Fifty-five per cent of mosquitoes approaching a house did not enter or made multiple attempts before passing through the eave. During approach, mosquitoes greatly reduced their speed and the flight paths became more convoluted. As a result, mosquitoes that passed through the eave spent more than 80 % of the observed time within 30 cm of the eave. Mosquitoes that exited the eave departed at eave level and followed the edge of the roof (12.5 %) or quickly re-entered after exiting (9.6 %)., Conclusions: The study shows that host-seeking mosquitoes, when entering a house, approach the eave in a wide angle to the house at eave level. Less than 25 % of approaching mosquitoes entered the house without interruption, whereas 12.5 % of mosquitoes that had entered left the house again within the time of observation. Advances in tracking techniques open a new array of questions that can now be answered to improve household interventions that combat malaria transmission.

The tick Ixodes ricinus is the main vector of the spirochaete Borrelia burgdorferi sensu lato, the causal agent of Lyme borreliosis, in the western Palearctic. Rodents are the reservoir host of B. afzelii, which can be transmitted to I. ricinus larvae during a blood meal. The infected engorged larvae moult into infected nymphs, which can transmit the spirochaetes to rodents and humans. Interestingly, even though only about 1% of the larvae develop into a borreliae-infected nymph, the enzootic borreliae lifecycle can persist. The development from larva to infected nymph is a key aspect in this lifecycle, influencing the density of infected nymphs and thereby Lyme borreliosis risk. The density of infected nymphs varies temporally and geographically and is influenced by multi-trophic (tick-host-borreliae) interactions. For example, blood feeding success of ticks and spirochaete transmission success differ between rodent species and host-finding success appears to be affected by a B. afzelii infection in both the rodent and the tick. In this paper, we review the major interactions between I. ricinus, rodents and B. afzelii that influence this development, with the aim to elucidate the critical factors that determine the epidemiological risk of Lyme borreliosis. The effects of the tick, rodent and B. afzelii on larval host finding, larval blood feeding, spirochaete transmission from rodent to larva and development from larva to nymph are discussed. Nymphal host finding, nymphal blood feeding and spirochaete transmission from nymph to rodent are the final steps to complete the enzootic B. afzelii lifecycle and are included in the review. It is concluded that rodent density, rodent infection prevalence, and tick burden are the major factors affecting the development from larva to infected nymph and that these interact with each other. We suggest that the B. afzelii lifecycle is dependent on the aggregation of ticks among rodents, which is manipulated by the pathogen itself. Better understanding of the processes involved in the development and aggregation of ticks results in more precise estimates of the density of infected nymphs, and hence predictions of Lyme borreliosis risk.

Malaria continues to place a disease burden on millions of people throughout the tropics, especially in sub-Saharan Africa. Although efforts to control mosquito populations and reduce human-vector contact, such as long-lasting insecticidal nets and indoor residual spraying, have led to significant decreases in malaria incidence, further progress is now threatened by the widespread development of physiological and behavioural insecticide-resistance as well as changes in the composition of vector populations. A mosquito-directed push-pull system based on the simultaneous use of attractive and repellent volatiles offers a complementary tool to existing vector-control methods. In this study, the combination of a trap baited with a five-compound attractant and a strip of net-fabric impregnated with micro-encapsulated repellent and placed in the eaves of houses, was tested in a malaria-endemic village in western Kenya. Using the repellent delta-undecalactone, mosquito house entry was reduced by more than 50%, while the traps caught high numbers of outdoor flying mosquitoes. Model simulations predict that, assuming area-wide coverage, the addition of such a push-pull system to existing prevention efforts will result in up to 20-fold reductions in the entomological inoculation rate. Reductions of such magnitude are also predicted when mosquitoes exhibit a high resistance against insecticides. We conclude that a push-pull system based on non-toxic volatiles provides an important addition to existing strategies for malaria prevention.

Background: The entomopathogenic fungus Metarhizium anisopliae shows great promise for the control of adult malaria vectors. A promising strategy for infection of mosquitoes is supplying the fungus at plant feeding sites., Methods: We evaluated the survival of fungus-exposed Anopheles gambiae mosquitoes (males and females) fed on 6% glucose and on sugars of Ricinus communis (Castor oil plant) and Parthenium hysterophorus (Santa Maria feverfew weed). Further, we determined the feeding propensity, quantity of sugar ingested and its digestion rate in the mosquitoes when fed on R. communis for 12 hours, one and three days post-exposure to fungus. The anthrone test was employed to detect the presence of sugar in each mosquito from which the quantity consumed and the digestion rates were estimated., Results: Fungus-exposed mosquitoes lived for significantly shorter periods than uninfected mosquitoes when both were fed on 6% glucose (7 versus 37 days), R. communis (7 versus 18 days) and P. hysterophorus (5 versus 7 days). Significantly fewer male and female mosquitoes, one and three days post-exposure to fungus, fed on R. communis compared to uninfected controls. Although the quantity of sugar ingested was similar between the treatment groups, fewer fungus-exposed than control mosquitoes ingested small, medium and large meals. Digestion rate was significantly slower in females one day after exposure to M. anisopliae compared to controls but remained the same in males. No change in digestion rate between treatments was observed three days after exposure., Conclusions: These results demonstrate that (a) entomopathogenic fungi strongly impact survival and sugar-feeding propensity of both sexes of the malaria vector An. gambiae but do not affect their potential to feed and digest meals, and (b) that plant sugar sources can be targeted as fungal delivery substrates. In addition, targeting males for population reduction using entomopathogenic fungi opens up a new strategy for mosquito vector control.

Background: The malaria mosquito Anopheles gambiae has a high preference for human hosts, a characteristic that contributes greatly to its capacity for transmitting human malaria. A sibling species, An. quadriannulatus, has a quite different host preference and feeds mostly on bovids. For this reason it does not contribute to human malaria transmission. Host seeking in mosquitoes is modulated by the olfactory system, which is primarily housed in the antennae and maxillary palps. Therefore, the detection of differing host odors by sibling species may be reflected in the expression level of the olfactory genes involved. Accordingly, we compared the transcriptomes of the antennae and maxillary palps of An. gambiae and An. quadriannulatus., Results: We identified seven relatively abundant olfactory receptors, nine ionotropic receptors and three odorant binding proteins that are substantially up-regulated in An. gambiae antennae. Interestingly, we find that the maxillary palps of An. gambiae contain a species-specific olfactory receptor, Or52, and five An. gambiae-specific gustatory receptors (AgGr48-52) that are relatively abundant. These five gustatory receptors are also expressed in An. gambiae antennae, although at lower level, indicating a likely role in olfaction, rather than gustation. We also document an approximately three-fold higher overall expression of olfaction genes in the maxillary palps of An. quadriannulatus, indicating an important role of this organ in the olfaction system of this species. Finally, the expression of the CO2 receptor genes is five to six-fold higher in the zoophilic An. quadriannulatus, implying a much higher sensitivity for detecting CO2., Conclusions: These results identify potential human host preference genes in the malaria vector An. gambiae. Interestingly, species-specific expression of several gustatory receptors in the olfactory organs indicate a role in olfaction rather than gustation. Additionally, a more expansive role for maxillary palps in olfaction is implicated than previously thought, albeit more so in the zoophilic An. quadriannulatus.

A strategy to decrease the vector competence of Anopheles gambiae sensu stricto (Diptera: Culicidae), the most efficient malaria vector in Africa, may consist of exploiting the genes involved in zoophily. Crossing and backcrossing experiments were performed between An. gambiae s.s. and the zoophilic sibling species Anopheles quadriannulatus. Mosquito strains were tested in a dual-choice olfactometer to investigate their responses to cow odour. Totals of 12% of An. gambiae s.s. and 59% of An. quadriannulatus selected the port with the cow odour. Crosses and backcrosses did not show a significant preference for the cow-baited port. The results indicated that anthropophilic behaviour in An. gambiae s.s. is a dominant or partially dominant trait, which, in conjunction with the unstable zoophilic behaviour observed in An. quadriannulatus, poses a serious obstacle to plans to decrease vector competence by modifying the anthropophilic trait., (© 2014 The Royal Entomological Society.)

Background: Monitoring of malaria vector populations provides information about disease transmission risk, as well as measures of the effectiveness of vector control. The Suna trap is introduced and evaluated with regard to its potential as a new, standardized, odour-baited tool for mosquito monitoring and control., Methods: Dual-choice experiments with female Anopheles gambiae sensu lato in a laboratory room and semi-field enclosure, were used to compare catch rates of odour-baited Suna traps and MM-X traps. The relative performance of the Suna trap, CDC light trap and MM-X trap as monitoring tools was assessed inside a human-occupied experimental hut in a semi-field enclosure. Use of the Suna trap as a tool to prevent mosquito house entry was also evaluated in the semi-field enclosure. The optimal hanging height of Suna traps was determined by placing traps at heights ranging from 15 to 105 cm above ground outside houses in western Kenya., Results: In the laboratory the mean proportion of An. gambiae s.l. caught in the Suna trap was 3.2 times greater than the MM-X trap (P < 0.001), but the traps performed equally in semi-field conditions (P = 0.615). As a monitoring tool , the Suna trap outperformed an unlit CDC light trap (P < 0.001), but trap performance was equal when the CDC light trap was illuminated (P = 0.127). Suspending a Suna trap outside an experimental hut reduced entry rates by 32.8% (P < 0.001). Under field conditions, suspending the trap at 30 cm above ground resulted in the greatest catch sizes (mean 25.8 An. gambiae s.l. per trap night)., Conclusions: The performance of the Suna trap equals that of the CDC light trap and MM-X trap when used to sample An. gambiae inside a human-occupied house under semi-field conditions. The trap is effective in sampling mosquitoes outside houses in the field, and the use of a synthetic blend of attractants negates the requirement of a human bait. Hanging a Suna trap outside a house can reduce An. gambiae house entry and its use as a novel tool for reducing malaria transmission risk will be evaluated in peri-domestic settings in sub-Saharan Africa.

Background: Pathogen transmission by mosquitos is known to be highly sensitive to mosquito bionomic parameters. Mosquito mark-release-recapture (MMRR) experiments are a standard method for estimating such parameters including dispersal, population size and density, survival, blood feeding frequency and blood meal host preferences., Methods: We assembled a comprehensive database describing adult female MMRR experiments. Bibliographic searches were used to build a digital library of MMRR studies and selected data describing the reported outcomes were extracted., Results: The resulting database contained 774 unique adult female MMRR experiments involving 58 vector mosquito species from the three main genera of importance to human health: Aedes, Anopheles and Culex. Crude examination of these data revealed patterns associated with geography as well as mosquito genus, consistent with bionomics varying by species-specific life history and ecological context. Recapture success varied considerably and was significantly different amongst genera, with 8, 4 and 1% of adult females recaptured for Aedes, Anopheles and Culex species, respectively. A large proportion of experiments (59%) investigated dispersal and survival and many allowed disaggregation of the release and recapture data. Geographic coverage was limited to just 143 localities around the world., Conclusions: This MMRR database is a substantial contribution to the compilation of global data that can be used to better inform basic research and public health interventions, to identify and fill knowledge gaps and to enrich theory and evidence-based ecological and epidemiological studies of mosquito vectors, pathogen transmission and disease prevention. The database revealed limited geographic coverage and a relative scarcity of information for vector species of substantial public health relevance. It represents, however, a wealth of entomological information not previously compiled and of particular interest for mosquito-borne pathogen transmission models.

Flight behavior of insecticide-resistant and susceptible malaria mosquitoes approaching deltamethrin-treated nets was examined using a wind tunnel. Behavior was linked to resulting health status (dead or alive) using comparisons between outcomes from free-flight assays and standard World Health Organization (WHO) bioassays. There was no difference in response time, latency time to reach the net, or spatial distribution in the wind tunnel between treatments. Unaffected resistant mosquitoes spent less time close to (< 30 cm) treated nets. Nettings that caused high knockdown or mortality in standard WHO assays evoked significantly less mortality in the wind tunnel; there was no excitorepellent effect in mosquitoes making contact with the nettings in free flight. This study shows a new approach to understanding mosquito behavior near insecticidal nets. The methodology links free-flight behavior to mosquito health status on exposure to nets. The results suggest that behavioral assays can provide important insights for evaluation of insecticidal effects on disease vectors., (© The American Society of Tropical Medicine and Hygiene.)

Background: Most odour baits for haematophagous arthropods contain carbon dioxide (CO2). The CO2 is sourced artificially from the fermentation of refined sugar (sucrose), dry ice, pressurized gas cylinders or propane. These sources of CO2 are neither cost-effective nor sustainable for use in remote areas of sub-Saharan Africa. In this study, molasses was evaluated as a potential substrate for producing CO2 used as bait for malaria mosquitoes., Methods: The attraction of laboratory-reared and wild Anopheles gambiae complex mosquitoes to CO2 generated from yeast-fermentation of molasses was assessed under semi-field and field conditions in western Kenya. In the field, responses of wild Anopheles funestus were also assessed. Attraction of the mosquitoes to a synthetic mosquito attractant, Mbita blend (comprising ammonia, L-lactic acid, tetradecanoic acid and 3-methyl-1-butanol) when augmented with CO2 generated from yeast fermentation of either molasses or sucrose was also investigated., Results: In semi-field, the release rate of CO2 and proportion of An. gambiae mosquitoes attracted increased in tandem with an increase in the quantity of yeast-fermented molasses up to an optimal ratio of molasses and dry yeast. More An. gambiae mosquitoes were attracted to a combination of the Mbita blend plus CO2 produced from fermenting molasses than the Mbita blend plus CO2 from yeast-fermented sucrose. In the field, significantly more female An. gambiae sensu lato mosquitoes were attracted to the Mbita blend augmented with CO2 produced by fermenting 500 g of molasses compared to 250 g of sucrose or 250 g of molasses. Similarly, significantly more An. funestus, Culex and other anopheline mosquito species were attracted to the Mbita blend augmented with CO2 produced from fermenting molasses than the Mbita blend with CO2 produced from sucrose. Augmenting the Mbita blend with CO2 produced from molasses was associated with high catches of blood-fed An. gambiae s.l. and An. funestus mosquitoes., Conclusion: Molasses is a suitable ingredient for the replacement of sucrose as a substrate for the production of CO2 for sampling of African malaria vectors and other mosquito species. The finding of blood-fed malaria vectors in traps baited with the Mbita blend and CO2 derived from molasses provides a unique opportunity for the study of host-vector interactions.

Background: Mosquito fitness is determined largely by body size and nutritional reserves. Plasmodium infections in the mosquito and resultant transmission of malaria parasites might be compromised by the vector's nutritional status. We studied the effects of nutritional stress and malaria parasite infections on transmission fitness of Anopheles mosquitoes., Methods: Larvae of Anopheles gambiae sensu stricto and An. stephensi were reared at constant density but with nutritionally low and high diets. Fitness of adult mosquitoes resulting from each dietary class was assessed by measuring body size and lipid, protein and glycogen content. The size of the first blood meal was estimated by protein analysis. Mosquitoes of each dietary class were fed upon a Plasmodium yoelii nigeriensis-infected mouse, and parasite infections were determined 5 d after the infectious blood meal by dissection of the midguts and by counting oocysts. The impact of Plasmodium infections on gonotrophic development was established by dissection., Results: Mosquitoes raised under low and high diets emerged as adults of different size classes comparable between An. gambiae and An. stephensi. In both species low-diet females contained less protein, lipid and glycogen upon emergence than high-diet mosquitoes. The quantity of larval diet impacted strongly upon adult blood feeding and reproductive success. The prevalence and intensity of P. yoelii nigeriensis infections were reduced in low-diet mosquitoes of both species, but P. yoelii nigeriensis impacted negatively only on low-diet, small-sized An. gambiae considering survival and egg maturation. There was no measurable fitness effect of P. yoelii nigeriensis on An. stephensi., Conclusions: Under the experimental conditions, small-sized An. gambiae expressed high mortality, possibly caused by Plasmodium infections, the species showing distinct physiological concessions when nutrionally challenged in contrast to well-fed, larger siblings. Conversely, An. stephensi was a robust, successful vector regardless of its nutrional status upon emergence. The data suggest that small-sized An. gambiae, therefore, would contribute little to malaria transmission, whereas this size effect would not affect An. stephensi.

Background: Malaria vector control is threatened by resistance to pyrethroids, the only class of insecticides used for treating bed nets. The second major vector control method is indoor residual spraying with pyrethroids or the organochloride DDT. However, resistance to pyrethroids frequently confers resistance to DDT. Therefore, alternative insecticides are urgently needed., Methodology/principal Findings: Insecticide resistance and the efficacy of indoor residual spraying with different insecticides was determined in a Gambian village. Resistance of local vectors to pyrethroids and DDT was high (31% and 46% mortality, respectively) while resistance to bendiocarb and pirimiphos methyl was low (88% and 100% mortality, respectively). The vectors were predominantly Anopheles gambiae s.s. with 94% of them having the putative resistant genotype kdr 1014F. Four groups of eight residential compounds were each sprayed with either (1) bendiocarb, a carbamate, (2) DDT, an organochlorine, (3) microencapsulated pirimiphos methyl, an organophosphate, or (4) left unsprayed. All insecticides tested showed high residual activity up to five months after application. Mosquito house entry, estimated by light traps, was similar in all houses with metal roofs, but was significantly less in IRS houses with thatched roofs (p=0.02). Residents participating in focus group discussions indicated that IRS was considered a necessary nuisance and also may decrease the use of long-lasting insecticidal nets., Conclusion/significance: Bendiocarb and microencapsulated pirimiphos methyl are viable alternatives for indoor residual spraying where resistance to pyrethroids and DDT is high and may assist in the management of pyrethroid resistance.

We determined the behavioral response of Aedes aegypti females to volatile compounds collected in indoor primary school classrooms. Volatiles were collected from classrooms from 0800 through 1030 h and 1130 through 1400 h in urban and rural schools in Tapachula, Chiapas, Mexico. Female responses to volatiles were assessed in a Y-tube olfactometer. Chemical compounds were identified using gas chromatography-mass spectrometer analysis. Volatiles from both schools were attractive when compared against their control. When such volatiles were compared, those from the rural school were more attractive than the ones from the urban school. Chromatographic profiles were similar between schools; however, the rural school showed more compounds. Attraction of Ae. aegypti females toward volatiles of primary school classrooms might increase dengue transmission probabilities in those sites.

Background: Different 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., Methods: Mosquitoes 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., Results: No 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., Conclusions: Both 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.

There is much evidence that some pathogens manipulate the behaviour of their mosquito hosts to enhance pathogen transmission. However, it is unknown whether this phenomenon exists in the interaction of Anopheles gambiae sensu stricto with the malaria parasite, Plasmodium falciparum--one of the most important interactions in the context of humanity, with malaria causing over 200 million human cases and over 770 thousand deaths each year. Here we demonstrate, for the first time, that infection with P. falciparum causes alterations in behavioural responses to host-derived olfactory stimuli in host-seeking female An. gambiae s.s. mosquitoes. In behavioural experiments we showed that P. falciparum-infected An. gambiae mosquitoes were significantly more attracted to human odors than uninfected mosquitoes. Both P. falciparum-infected and uninfected mosquitoes landed significantly more on a substrate emanating human skin odor compared to a clean substrate. However, significantly more infected mosquitoes landed and probed on a substrate emanating human skin odor than uninfected mosquitoes. This is the first demonstration of a change of An. gambiae behaviour in response to olfactory stimuli caused by infection with P. falciparum. The results of our study provide vital information that could be used to provide better predictions of how malaria is transmitted from human being to human being by An. gambiae s.s. females. Additionally, it highlights the urgent need to investigate this interaction further to determine the olfactory mechanisms that underlie the differential behavioural responses. In doing so, new attractive compounds could be identified which could be used to develop improved mosquito traps for surveillance or trapping programmes that may even specifically target P. falciparum-infected An. gambiae s.s. females.

Mosquitoes use plant sugars and vertebrate blood as nutritional resources. When searching for blood hosts, some mosquitoes express preferential behavior for selected species. Here, we review the available knowledge on host preference, as this is expected to affect the life history and transmission of infectious pathogens. Host preference is affected by myriad extrinsic and intrinsic factors. Inherent factors are determined by genetic selection, which appears to be controlled by adaptive advantages that result from feeding on certain host species. Host preference of mosquitoes, although having a genetic basis, is characterized by high plasticity mediated by the density of host species, which by their abundance form a readily accessible source of blood. Host-selection behavior in mosquitoes is an exception rather than the rule. Those species that express strong and inherent host-selection behavior belong to the most important vectors of infectious diseases, which suggests that this behavioral trait may have evolved in parallel with parasite-host evolution.

Background: Control of mosquitoes that transmit malaria has been the mainstay in the fight against the disease, but alternative methods are required in view of emerging insecticide resistance. Entomopathogenic fungi are candidate alternatives, but to date, few trials have translated the use of these agents to field-based evaluations of their actual impact on mosquito survival and malaria risk. Mineral oil-formulations of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were applied using five different techniques that each exploited the behaviour of malaria mosquitoes when entering, host-seeking or resting in experimental huts in a malaria endemic area of rural Tanzania., Results: Survival of mosquitoes was reduced by 39-57% relative to controls after forcing upward house-entry of mosquitoes through fungus treated baffles attached to the eaves or after application of fungus-treated surfaces around an occupied bed net (bed net strip design). Moreover, 68 to 76% of the treatment mosquitoes showed fungal growth and thus had sufficient contact with fungus treated surfaces. A population dynamic model of malaria-mosquito interactions shows that these infection rates reduce malaria transmission by 75-80% due to the effect of fungal infection on adult mortality alone. The model also demonstrated that even if a high proportion of the mosquitoes exhibits outdoor biting behaviour, malaria transmission was still significantly reduced., Conclusions: Entomopathogenic fungi strongly affect mosquito survival and have a high predicted impact on malaria transmission. These entomopathogens represent a viable alternative for malaria control, especially if they are used as part of an integrated vector management strategy.

Background: Rice paddies harbour a large variety of organisms including larvae of malaria mosquitoes. These paddies are challenging for mosquito control because their large size, slurry and vegetation make it difficult to effectively apply a control agent. Aquatain, a monomolecular surface film, can be considered a suitable mosquito control agent for such breeding habitats due to its physical properties. The properties allow Aquatain to self-spread over a water surface and affect multiple stages of the mosquito life cycle., Methodology/principal Findings: A trial based on a pre-test/post-test control group design evaluated the potential of Aquatain as a mosquito control agent at Ahero rice irrigation scheme in Kenya. After Aquatain application at a dose of 2 ml/m(2) on rice paddies, early stage anopheline larvae were reduced by 36%, and late stage anopheline larvae by 16%. However, even at a lower dose of 1 ml/m(2) there was a 93.2% reduction in emergence of anopheline adults and 69.5% reduction in emergence of culicine adults. No pupation was observed in treated buckets that were part of a field bio-assay carried out parallel to the trial. Aquatain application saved nearly 1.7 L of water in six days from a water surface of 0.2 m(2) under field conditions. Aquatain had no negative effect on rice plants as well as on a variety of non-target organisms, except backswimmers., Conclusions/significance: We demonstrated that Aquatain is an effective agent for the control of anopheline and culicine mosquitoes in irrigated rice paddies. The agent reduced densities of aquatic larval stages and, more importantly, strongly impacted the emergence of adult mosquitoes. Aquatain also reduced water loss due to evaporation. No negative impacts were found on either abundance of non-target organisms, or growth and development of rice plants. Aquatain, therefore, appears a suitable mosquito control tool for use in rice agro-ecosystems.

Increased human population in the Western Kenya highlands has led to reclamation of natural swamps resulting in the creation of habitats suitable for the breeding of Anopheles gambiae, the major malaria vector in the region. Here we report on a study to restore the reclaimed swamp and reverse its suitability as a habitat for malaria vectors. Napier grass-shaded and non-shaded water channels in reclaimed sites in Western Kenya highlands were studied for the presence and density of mosquito larvae, mosquito species composition, and daily variation in water temperature. Shading was associated with 75.5% and 88.4% (P < 0.0001) reduction in anopheline larvae densities and 78.1% and 88% (P < 0.0001) reduction in Anopheles gambiae sensu lato (s.l.) densities in two sites, respectively. Shading was associated with a 5.7°C, 5.0°C, and 4.7°C, and 1.6°C, 3.9°C, and 2.8°C (for maximum, minimum, and average temperatures, respectively) reduction (P < 0.0001) in water temperatures in the two locations, respectively. An. gambiae s.l. was the dominant species, constituting 83.2% and 73.1%, and 44.5% and 42.3%, of anophelines in non-shaded and shaded channels, respectively, in the two sites, respectively. An. gambiae sensu stricto (s.s.) constituted the majority (97.4%) of An. gambiae s.l., while the rest (2.6%) comprised of Anopheles arabiensis. Minimum water temperature decreased with increasing grass height (P = 0.0039 and P = 0.0415 for Lunyerere and Emutete sites, respectively). The results demonstrate how simple environmental strategies can have a strong impact on vector densities.

Background: The relationship between mosquito development and temperature is one of the keys to understanding the current and future dynamics and distribution of vector-borne diseases such as malaria. Many process-based models use mean air temperature to estimate larval development times, and hence adult vector densities and/or malaria risk., Methods: Water temperatures in three different-sized water pools, as well as the adjacent air temperature in lowland and highland sites in western Kenya were monitored. Both air and water temperatures were fed into a widely-applied temperature-dependent development model for Anopheles gambiae immatures, and subsequently their impact on predicted vector abundance was assessed., Results: Mean water temperature in typical mosquito breeding sites was 4-6 degrees C higher than the mean temperature of the adjacent air, resulting in larval development rates, and hence population growth rates, that are much higher than predicted based on air temperature. On the other hand, due to the non-linearities in the relationship between temperature and larval development rate, together with a marginal buffering in the increase in water temperature compared with air temperature, the relative increases in larval development rates predicted due to climate change are substantially less., Conclusions: Existing models will tend to underestimate mosquito population growth under current conditions, and may overestimate relative increases in population growth under future climate change. These results highlight the need for better integration of biological and environmental information at the scale relevant to mosquito biology.

Background: Ideally larval control activities should be targeted at sites that generate the most adult vectors, thereby reducing operational costs. Despite the plethora of potential mosquito breeding sites found in the floodplains of the Gambia River, about 150 km from its mouth, during the rainy season, only a small proportion are colonized by anophelines on any day. This study aimed to determine the characteristics of larval habitats most frequently and most densely populated by anopheline larvae and to estimate the numbers of adults produced in different habitats., Methods: A case-control design was used to identify characteristics of sites with or without mosquitoes. Sites were surveyed for their physical water properties and invertebrate fauna. The characteristics of 83 sites with anopheline larvae (cases) and 75 sites without (controls) were collected between June and November 2005. Weekly adult productivity was estimated with emergence traps in water-bodies commonly containing larvae., Results: The presence of anopheline larvae was associated with high invertebrate diversity (Odds Ratio, OR 11.69, 95% CI 5.61-24.34, p < 0.001), the presence of emergent vegetation (OR 2.83, 95% CI 1.35-5.95, p = 0.006), and algae (at borderline significance; OR 1.87, 95% CI 0.96-3.618, p = 0.065). The density of larvae was reduced in sites that were larger than 100 m in perimeter (OR 0.151; 95% CI 0.060-0.381, p < 0.001), where water was tidal (OR 0.232; 95% CI 0.101-0.533, p = 0.001), vegetation shaded over 25% of the habitat (OR 0.352; 95% CI 0.136-0.911, p = 0.031) and water conductivity was above 2,000 muS/cm (OR 0.458; 95% CI 0.220-0.990, p = 0.048). Pools produced the highest numbers of Anopheles gambiae adults compared with rice fields, floodwater areas close to the edge of the floodplain or close to the river, and stream fringes. Pools were characterized by high water temperature and turbidity, low conductivity, increased presence of algae, and absence of tidal water., Conclusion: There are few breeding sites that produce a high number of adult vectors in the middle reaches of the river in The Gambia, whereas those with low productivity are larger in area and can be found throughout the rainy season. Even though risk factors could be identified for the presence and density of larvae and productivity of habitats, the results indicate that anti-larval interventions in this area of The Gambia cannot be targeted in space or time during the rainy season.

Since 2005, Aedes albopictus mosquitoes have been detected in companies in the Netherlands that import ornamental plants from China. To assess the risks of dengue transmission, a study was carried out in 48 persons who are professionally exposed to these mosquitoes. No evidence for non-travel-related flavivirus infections was found.

Initiatives on integrated vector management (IVM) approaches are increasingly undertaken as alternatives to existing vector control. An impact model of IVM is presented with performance and impact indicators at six causal steps from coverage of the intervention to impact on disease. Impacts in fields other than health are also discussed because of the emphasis in IVM on capacity building, partnerships and sustainability. A conceptual framework for evaluation of IVM is designed, based on considerations of the selection of indicators, level of inference, cluster size and method of evaluation. The framework, which is tested in three case studies, is intended as guidance for public health workers and policy-makers.

After bluetongue (BT) appeared in northern Europe in August 2006 entomological studies were implemented in all five affected Member States (MSs) to establish which species of Culicoides had acted as vectors. The findings can be summarised as follows: (i) C. imicola the principal southern European/African vector of BTV has not penetrated into northern Europe, (ii) three pools of C. obsoletus/C. scoticus and one of C. dewulfi assayed RT-PCR-positive to BTV-8, (iii) in support of these results it was found that both potential vectors had also high parity rates (approximately 40%) indicating increased longevity favouring BTV virogenesis and transmission, (iv) furthermore, C. obsoletus/C. scoticus and C. dewulfi occurred also widely and abundantly on sheep and cattle holdings across the entire affected region, (v) and during the latter part of the season showed strong endophily readily entering livestock buildings in significant numbers to bite the animals inside (endophagy), (vi) which demonstrates that housing at best offers only limited protection to livestock from Culicoides attacks, (vii) in contrast the potential vector C. pulicaris sensu stricto was restricted geographically, was captured rarely, had a low parity rate (10%) and was exophilic indicating it played no role in the outbreak of BT, (viii) the incrimination of C. dewulfi as a novel vector is significant because it breeds in cattle and horse dung this close association raising its vectorial potential, but (ix) problems with its taxonomy (and that of the Obsoletus and Pulicaris species complexes) illustrates the need for morphological and molecular techniques to become more fully integrated to ensure progress in the accurate identification of vector Culicoides, (x) midge densities (as adjudged by light traps) were generally low indicating northern European Culicoides to have a high vector potential and/or that significant numbers of midges are going undetected because they are biting (and transmitting BTV) during the day when light traps are not effective, and (xi) the sporadic capture of Culicoides in the winter of 2007 invites re-examination of the current definition of a vector-free period. The re-emergence of BT over a wide front in 2007 raises anew questions as to precisely how the virus overwinters and asks also that we scrutinise our monitoring systems in terms of their sensitivity and early warning capability.

The Netherlands has enjoyed a relatively free state of vector-borne diseases of economic importance for more than one century. Emerging infectious diseases may change this situation, threatening the health of humans, domestic livestock and wildlife. In order to be prepared for the potential outbreak of vector-borne diseases, a study was undertaken to investigate the distribution and seasonal dynamics of candidate vectors of infectious diseases with emphasis on bluetongue vectors (Culicoides spp.). The study focused primarily on the relationship between characteristic ecosystems suitable for bluetongue vectors and climate, as well as on the phenology and population dynamics of these vectors. Twelve locations were selected, distributed over four distinct habitats: a wetland area, three riverine systems, four peat land areas and four livestock farms. Culicoides populations were sampled continuously using CO(2)-baited counterflow traps from July 2005 until August 2006, with an interruption from November 2005 to March 2006. All vectors were identified to species level. Meteorological and environmental data were collected at each location. Culicoides species were found in all four different habitat types studied. Wetland areas and peat bogs were rich in Culicoides spp. The taxonomic groups Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) were strongly associated with farms. Eighty-eight percent of all Culicoides consisted of the taxon C. obsoletus/Culicoides scoticus. On the livestock farms, 3% of Culicoides existed of the alleged bluetongue vector Culicoides dewulfi Goetghebuer. Culicoides impunctatus Goetghebuer was strongly associated with wetland and peat bog. Many Culicoides species were found until late in the phenological season and their activity was strongly associated with climate throughout the year. High annual variations in population dynamics were observed within the same study areas, which were probably caused by annual variations in environmental conditions. The study demonstrates that candidate vectors of bluetongue virus are present in natural and livestock-farm habitats in the Netherlands, distributed widely across the country. Under favourable climatic conditions, following virus introduction, bluetongue can spread among livestock (cattle, sheep and goats), depending on the nature of the viral serotype. The question now arises whether the virus can survive the winter conditions in north-western Europe and whether measures can be taken that effectively halt further spread of the disease.

We studied the use of African water storage pots for point source application of Metarhizium anisopliae against the malaria vectors Anopheles gambiae s.s. and An. funestus. Clay pots were shown to be attractive resting sites for male and female An. gambiae s.s. and were not repellent after impregnation with fungus. M. anisopliae was highly infective and virulent after spray application inside pots. At a dosage of 4 x 10(10) conidia/m(2), an average of 95 +/- 1.2% of An. gambiae s.s. obtained a fungal infection. A lower dosage of 1 x 10(10) conidia/m(2) infected an average of 91.5 +/- 0.6% of An. gambiae s.s. and 91.8 +/- 1.2% of An. funestus mosquitoes. Fungal infection significantly reduced mosquito longevity, as shown by differences between survival curves and LT(50) values. These pots are suitable for application of entomopathogenic fungi against malaria vectors and their potential for sustainable field implementation is discussed.

Integrated vector management (IVM) aims to extend the basis of disease control by involving other sectors and local communities in control action. It is vital that decisions on IVM are made locally for two reasons: first, the epidemiology of disease can vary at a small spatial scale, suggesting the need for precise targeting and second, a number of disease determinants relate to the actions and conditions of local communities, suggesting the need for their increased participation. This requires a shift from centrally managed, sector-specific operations to the facilitation of multi-partner programmes at the district and local level. We propose a methodology for involving local partners outside the health sector in describing and mapping the local determinants and conditions of disease, analysing control options, and consolidating a joint strategy of control. Thus determinants that often lie outside the domain of the health sector are tackled.

Background: Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO(2)) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown., Results: Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO(2)-responsive and characterized by the coexpression of three receptors that confer CO(2) responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses., Conclusions: Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.

Several highland areas in eastern Africa have recently suffered from serious malaria epidemics. Some models predict that, in the short term, these areas will experience more epidemics as a result of global warming. However, the various processes underlying these changes are poorly understood. We therefore investigated malaria prevalence, malaria vector densities and malaria vector survival in a highland area in western Kenya, ranging from approximately 1,550-1,650 m altitude. Although only five adult malaria vectors were collected during 180 light traps and 180 resting collections over a 23-month study period, malaria was prevalent among school children (average parasite prevalence: 10%). During an extensive survey of potential larval habitats, we identified only seven habitats containing Anopheles gambiae Giles s.l. larvae. Their limited number and low larval densities suggested that their contribution to the adult vector population was small. Experiments on adult and larval survival showed that at this altitude, adult mosquitoes survived inside local houses, but that larval development was severely retarded: only 2 of 500 A. gambiae s.l. larvae developed to the pupal stage, whereas all other larvae died prior to pupation. At present, high vector densities are unlikely because of unfavourable abiotic conditions in the area. However, temporary favourable conditions, such as during El Niño years, may increase larval vector survival and may lead to malaria epidemics.

Background: The successful development of odour-baited trapping systems for mosquitoes depends on the identification of behaviourally active semiochemicals, besides the design and operating principles of such devices. A large variety of 'attractants' has been identified in laboratory investigations, yet few of these increase trap catches in the field. A contained system, intermediate between the laboratory and open field, is presented and previous reports that human foot odour induces behavioural responses of Anopheles gambiae confirmed., Methods: The response of 3-5 day old female An. gambiae towards odour-baited counterflow geometry traps (MM-X model; American Biophysics Corp., RI) was studied in semi-field (screen house) conditions in western Kenya. Traps were baited with human foot odour (collected on socks), carbon dioxide (CO2, 500 ml min(-1)), ammonia (NH3), 1-octen-3-ol, or various combinations thereof. Trap catches were log (x+1) transformed and subjected to Latin square analysis of variance procedures., Results: Apart from 1-octen-3-ol, all odour baits caused significant (P < 0.05) increases in trap catches over non-baited traps. Foot odour remained behaviourally active for at least 8 days after collection on nylon or cotton sock fabric. A synergistic response (P < 0.001) was observed towards the combination of foot odour and CO2, which increased catches of these odours alone by 3.8 and 2.7 times, respectively., Conclusion: These results are the first to report behavioural responses of an African malaria vector to human foot odour outside the laboratory, and further investigation of fractions and/or individual chemical components of this odour complex are called for. Semi-field systems offer the prospect of high-throughput screening of candidate kairomones, which may expedite the development of efficient trap-bait systems for this and other African mosquito species.

The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent against adult African malaria vectors. In addition to causing significant mortality, this pathogen is known to cause reductions in feeding and fecundity in a range of insects. In the present study we investigated whether infection with M. anisopliae affected blood feeding and fecundity of adult female malaria vectors Anopheles gambiae Giles sensu stricto. Mosquitoes were contaminated with either a low or a moderately high dose of oil-formulated conidia of M. anisopliae, and offered a single human blood meal 48, 72, or 96 h later to assess feeding propensity and individual blood meal size. In a second experiment, individual fungus-infected females were offered a blood meal every third day (to a total of 8 gonotrophic cycles), and allowed to oviposit after each cycle in order to quantify feeding propensity and fecundity. Infected females took smaller blood meals and displayed reduced feeding propensity. It was found that mosquitoes, inoculated with a moderately high dose of fungal conidia, exhibited reduced appetite related to increasing fungal growth. Of the fungus-infected females, the proportion of mosquitoes taking the second blood meal was reduced with 51%. This was further reduced to 35.3% by the 4th blood meal. During 8 feeding opportunities, the average number of blood meals taken by uninfected females was 4.39, against 3.40 (low dose), and 2.07 (high dose) blood meals for the fungus-infected females. Moreover, infected females produced fewer eggs per gonotrophic cycle and had a lower life-time fecundity. Epidemiological models show that both blood feeding and fecundity are among the most important factors affecting the likelihood of a mosquito transmitting malaria, which suggests that this fungus may have potential as biocontrol agent for vector-borne disease control.

Biological control of malaria mosquitoes in Africa has rarely been used in vector control programs. Recent developments in this field show that certain fungi are virulent to adult Anopheles mosquitoes. Practical delivery of an entomopathogenic fungus that infected and killed adult Anopheles gambiae, Africa's main malaria vector, was achieved in rural African village houses. An entomological inoculation rate model suggests that implementation of this vector control method, even at the observed moderate coverage during a field study in Tanzania, would significantly reduce malaria transmission intensity.

Background: The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides., Methods: Virgin female Anopheles gambiae sensu stricto were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity., Results: Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 +/- 3.2% and 33.3 +/- 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males (P < 0.05)., Conclusion: This study shows that autodissemination of fungal inoculum between An. gambiae s.s. mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.

Background: Although the effects of rainfall on the population dynamics of the malaria vector Anopheles gambiae have been studied in great detail, the effects of dry periods on its survival remain less clear., Methods: The effects of drying conditions were simulated by creating desiccated habitats, which consisted of trays filled with damp soil. Experiments were performed in these trays to (i) test the ability of An. gambiae sensu stricto eggs to hatch on damp soil and for larvae to reach an artificial breeding site at different distances of the site of hatching and (ii) to record survival of the four larval stages of An. gambiae s.s. when placed on damp soil., Results: Eggs of An. gambiae s.s. hatched on damp soil and emerging larvae were capable of covering a distance of up to 10 cm to reach surface water enabling further development. However, proportions of larvae reaching the site decreased rapidly with increasing distance. First, second and third-instar larvae survived on damp soil for an estimated period of 64, 65 and 69 hrs, respectively. Fourth-instar larvae survived significantly longer and we estimated that the maximum survival time was 113 hrs., Conclusion: Short-term survival of aquatic stages of An. gambiae on wet soil may be important and adaptive when considering the transient nature of breeding sites of this species in sub-Saharan Africa. In addition, the results suggest that, for larval vector control methods to be effective, habitats should remain drained for at least 5 days to kill all larvae (e.g. in rice fields) and habitats that recently dried up should be treated as well, if larvicidal agents are applied.

A field study was carried out in the large-scale rice irrigation scheme of the Office du Niger in Mali to investigate the relation between anopheline mosquito larval development and small-scale differences in irrigation practices, such as water level, irrigation application and irrigation frequency. The objective of the study was to find out if water management can be used as a tool for vector control to reduce the malaria transmission risk. Larvae of Anopheles gambiae s.s.,; the main malaria vector in the study area, developed mostly in the first 6 weeks after transplanting the rice. During rice development, a succession of anopheline species was observed. This was associated with a marked decrease in light intensity reaching the water surface as plant height increased. Minor differences in water management resulted in noticeable variations in larval densities and species composition. A. gambiae s.s. larvae were most abundant during the early growing stages and almost absent in a closed rice crop. Due to improper drainage after harvest, A. gambiae s.s. breeding was soon re-established in fields where small pools of water were retained. The results suggest that larval mosquito habitats in the Office du Niger can be significantly reduced by water management, simultaneous planting and harvesting and proper drainage of fallow fields., (Copyright 2002 Elsevier Science B.V.)

Background: Many studies have suggested that variability in the attractiveness of humans to host-seeking mosquitoes is caused by differences in the make-up of body emanations, and olfactory signals in particular. Most investigations have either been laboratory-based, utilising odour obtained from sections of the body, or have been done in the field with sampling methods that do not discriminate between visual, physical and chemical cues of the host. Accordingly, evidence for differential attractiveness based on body emanations remains sparse in spite of the far-reaching epidemiological implications of this phenomenon., Methods: A new three-port olfactometer that accommodates complete human beings as sources of host-seeking stimuli was used to study behavioural responses of Anopheles gambiae Giles sensu stricto (hereafter An. gambiae) under semi-field conditions in western Kenya. Differential attractiveness of nine male Kenyans was assessed by simultaneously exposing the mosquitoes to (a mixture of) total body emanations of 3 people occupying separate tents. Controls (empty tents) were included and the effect of residual odours following tent occupation was also examined., Results: Trap catches increased significantly (P < 0.001) when a tent was occupied. Based on 'competition' experiments, the nine persons were classified into least, medium and most attractive groups. There was no significant interaction between person and trap (P = 0.302) or person and test period (P = 0.223). Presence (P < 0.001) or absence (P = 0.949) of significant differences in the number of mosquitoes caught per trap when tents were simultaneously occupied by one person in each or left empty, respectively, demonstrated that residual odours following tent occupation did not affect behavioural responses of the mosquitoes., Conclusion: We provide evidence that in the vicinity of humans, when exposed to a blend of physical and olfactory signals from more than one host, An. gambiae can effectively and consistently express host-selection behaviour that results in non-random biting.

Little is known about the transmission dynamics of human malaria and other vector-borne diseases, partly because of the limited availability and distribution of appropriate tools for quantifying human-mosquito contact rates. Recent developments in molecular biology have allowed a significant increase in the efficacy and reliability of bloodmeal identification, and DNA-based molecular markers are now being harnessed for typing arthropod bloodmeals. The extent to which these markers have been used for analysis of mosquito bloodmeals and the potential they might have for the future is discussed, and the contributions that the advent of PCR has made are examined here.

Ecological and population biology issues constitute serious challenges to the application of genetically modified mosquitos (GMM) for disease control.

Anopheles quadriannulatus, the cattle-feeding member of the An. gambiae complex, was fed human blood which contained cultured gametocytes of Plasmodium falciparum, using a membrane feeding system. After 7 days, 33-80% of the mosquitoes that took a blood meal contained developing oocysts. In 7 out of 12 females sporozoites were found in the salivary glands 14 days after the infectious blood meal. Control groups of An. gambiae s.s. and An. stephensi became readily infected with > 90% developing oocysts. All of the An. gambiae dissected 14 days after the infectious blood meal had sporozoites in their salivary glands. The results demonstrate that An. quadriannulatus is susceptible to infections with P. falciparum.

Attempts are being made to backcross into Anopheles gambiae s.s. the gene(s) which cause zoophily in Anopheles quadriannulatus. Such a backcrossed strain might be preferable to a Plasmodium-refractory strain as a basis for genetic control because a refractory strain could select for evasion of refractoriness in the wild Plasmodium population. The species composition of the malaria vector population in several Tanzanian villages was overwhelmingly An. gambiae s.s. in a normal rainy season, but consisted of four species, all proved by ELISA and/or PCR to carry P. falciparum sporozoites, at the time of the heavy rains associated with El Niño. Thus any scheme, for malaria transmission control by replacement of vectors by genetically-manipulated non-vectors, would have to be able to replace more than one species.

Small scale spatial variation and temporal heterogeneity in mosquito densities can have important consequences for disease transmission, but the extreme variation which is observed in populations of malaria vectors makes it difficult to obtain good predictions of densities for short time periods over limited areas. We have applied Bayesian techniques derived for use in cancer epidemiology in order to map densities of Anopheles gambiae s.l. and A. funestus in a Tanzanian village where there is intense transmission of Plasmodium falciparum malaria. Estimates derived in this way should prove useful in vector population biology and in improving estimates of exposure-response relationships of the human host to malaria. The same methods can be applied in other fields of animal ecology.